Macromolecules comprising a thioether cross-link

ABSTRACT

The present invention provides macromolecules comprising at least one thioether cross-link. A thioether cross-link comprising a single thioether bond between two residues of a macromolecule. The macromolecules of the invention can display enhanced stability, pharmaceutical properties and functional properties. In particular, the invention provides an isolated antibodies comprising at least one thioether cross-link that specifically bind to particular antigens. The present invention also provides a composition comprising a macromolecule substantially free of a denaturing reagent, wherein the macromolecule comprises at least one thioether cross-link. In addition, the present invention provides a method for producing the macromolecules and compositions of the invention.

This application claims the benefit of priority of U.S. provisionalapplication Nos. 60/661,724 and 60/699,138, filed Mar. 14, 2005, andJul. 13, 2005, respectively, the contents of which are herebyincorporated by reference in their entireties.

1. FIELD OF THE INVENTION

The present invention provides compositions comprising macromolecules,for instance, polypeptides, e.g., an antibody, that comprise at leastone thioether cross-link. This novel class of macromolecules can displayenhanced stability, pharmaceutical properties and functional properties.The invention also provides isolated antibodies comprising at least onethioether cross-link that specifically bind to particular antigens. Inaddition, the present invention provides methods for producing themacromolecules and compositions of the invention.

2. BACKGROUND OF THE INVENTION

Antibodies play a vital role in our immune response. They can inactivateviruses and bacterial toxins, and are essential in recruiting thecomplement system and various types of white blood cells to killinvading microorganisms and large parasites. Antibodies are synthesizedexclusively by B lymphocytes and are produced in millions of forms, eachwith a different amino acid sequence and a different binding site for anantigen.

A typical antibody is a Y-shaped molecule with two identical heavy (H)chains (each containing about 440 amino acids) and two identical light(L) chains (each containing about 220 amino acids). Proteolytic enzymes,such as papain and pepsin, can split an antibody molecule into differentcharacteristic fragments. Papain produces two separate and identical Fabfragments, each with one antigen-binding site, and one Fc fragment.Pepsin produces one F(ab′)₂ fragment. Alberts et al., Molecular Biologyof the Cell, 2nd ed., 1989, Garland Publishing, Inc.

Both L and H chains have a variable sequence at their amino-terminalends but a constant sequence at their carboxyl-terminal ends. The Lchains have a constant region of about 110 amino acids long and avariable region of the same size. The H chains also have a variableregion of about 110 amino acids long, but the constant region of the Hchains is about 330 or 440 amino acid long, depending on the class ofthe H chain. Alberts et al., Molecular Biology of the Cell, 2nd ed.,1989, Garland Publishing, Inc.

The association between the four chains involves both covalent andnoncovalent interactions. The covalent interactions are disulfide bondsformed between the cysteine residues in the carboxyl terminus of thelight chain and the C_(H) 1 domain of the heavy chain and disulfidebonds formed between the cysteine residues in the hinge regions of thetwo heavy chains.

Natural immunoglobulins have been used in assays, diagnosis and, to amore limited extent, therapy. However, such uses, especially in therapy,have been hindered by the polyclonal nature of natural immunoglobulins.The advent of monoclonal antibodies of defined specificity increased theopportunities for therapeutic use. Therapeutic monoclonal antibodiesusually contain some microheterogeneity resulting frompost-translational modifications and degradation events that occurduring the production process and throughout the shelf-life of thebiopharmaceutical product. The present invention provides a novelmodification of monoclonal antibodies.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention provides macromolecules comprising a thioethercross-link. In some embodiments, the macromolecule is a polypeptide,either a monomer or a multi-chain polypeptide. In further embodiments,the polypeptide is an antibody. The antibodies of the present inventionmay be, but are not limited to polyclonal antibodies, monoclonalantibodies, Fab fragments, F(ab)′ fragments, F(ab)′₂ fragments, singlechain antibodies, human antibodies, humanized or chimeric antibodies,and epitope-binding fragments of any of the above.

Briefly, a thioether cross-link is a thioether comprising a singlethioether bond between two residues of a macromolecule. In certainembodiments, the thioether cross-links have a structure according toformula I: —OOC—CH(NH—)—R¹—S—R²—CH(NH—)—COO—, wherein R¹ and R² areindependently side chains of macromolecular residues, such as amino acidresidues. Preferred R¹ and R² groups include methylene, ethylene,propylene, and butylene. Particularly preferred is methylene.

In one aspect, the present invention provides a compositionsubstantially free of a denaturing reagent comprising a polypeptide,wherein the polypeptide comprises at least one thioether cross-link. Insome embodiments, the polypeptide is more than 2%, 5%, 10% or 15% of thepolypeptide molecules in the composition. In certain embodiments, thepolypeptide is an antibody.

The thioether cross-link can link any two residues of a polypeptide orof two polypeptide chains. For example, in certain embodiments, theresidues involved in a thioether cross-link can be any pair of thefollowing: a cysteine, aspartic acid, glutamic acid, histidine,methionine, tyrosine, or any other naturally or non-naturally occurringamino acid. In certain embodiments, the thioether links a cysteineresidue of the heavy chain and a cysteine residue of the light chain.The cysteine residue can be in any region of the heavy chain or lightchain. In certain embodiments, the cysteine residue of the heavy chainis in the C_(H)1 region and the cysteine residue of the light chain isin C_(L) region. In one embodiment, the cysteine residue in the C_(H)1region of the heavy chain is at the amino acid position 223 according tothe Kabat numbering system, and the cysteine residue in the C_(L) regionof the heavy chain is at the amino acid position 213 according to theKabat numbering system.

The thioether cross-link can be at any location of a macromolecule. Forinstance, the thioether cross-link can be inter-molecular orintra-molecular. In some embodiments, the thioether cross-link isintra-molecular. For example, the thioether cross-link can link twopolypeptide chains of an antibody. In some embodiments, the thioethercross-link links a heavy chain and a light chain of the antibody.

The thioether cross-link can be located in any position of apolypeptide. For example, the thioether cross-link can be at theN-terminus, at the C-terminus, or between the N-terminus and C-terminusof a polypeptide chain. In some embodiments, the thioether cross-link islocated between the N-terminus and C-terminus of a polypeptide chain.

In certain embodiments, the present invention provides a compositioncomprising an antibody or fragment thereof, wherein the antibody orfragment thereof comprises at least one thioether cross-link and whereinthe antibody or fragment thereof specifically binds to one or moreparticular antigens.

In certain embodiments, the antibody of the present inventionspecifically binds to an antigen of respiratory syncytial virus (RSV).In some embodiments, the antibody comprises the amino acid sequence ofthe variable heavy (V_(H)) and variable light (V_(L)) chains ofpalivizumab or motavizumab. In other embodiments, the antibody comprisesthe amino acid sequence of the complementarity determining regions(CDRs) of the V_(H) and V_(L) chains of palivizumab or motavizumab.

In certain embodiments, the antibody of the present inventionspecifically binds to an antigen of human metapneumovirus (hMPV). Insome embodiments, the antibody is a humanized antibody that specificallybinds to an antigen of hMPV.

In certain embodiments, the antibody of the present inventionspecifically binds to integrin α_(v)β₃. In some embodiments, theantibody comprises the amino acid sequence of the V_(H) and V_(L) chainsof MEDI-522 (Vitaxin®). In other embodiments, the antibody comprises theamino acid sequence of the CDRs of the V_(H) and V_(L) chains ofMEDI-522 (Vitaxin®).

In certain embodiments, the antibody of the present inventionspecifically binds to CD2. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of siplizumab. Inother embodiments, the antibody comprises the amino acid sequence of thecomplementarity determining regions (CDRs) of V_(H) and V_(L) chains ofsiplizumab.

In certain embodiments, the antibody of the present inventionspecifically binds to CD19. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of MT103. In otherembodiments, the antibody comprises the amino acid sequence of the CDRsof the V_(H) and V_(L) chains of MT103.

In certain embodiments, the antibody of the present inventionspecifically binds to an Eph receptor. In certain embodiments, theantibody of the present invention specifically binds to EphA2. In someembodiments, the antibody comprises the amino acid sequence of the V_(H)and V_(L) chains of EA2 or EA5. In other embodiments, the antibodycomprises the amino acid sequence of the V_(H) and V_(L) chains of EA2or EA5. In certain embodiments, the antibody of the present inventionspecifically binds to EphA4. In some embodiments, the antibody of thepresent invention specifically binds to EphB4.

In certain embodiments, the antibody of the present inventionspecifically binds to IL-9. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of MEDI-528. Inother embodiments, the antibody comprises the amino acid sequence of theCDRs of the V_(H) and V_(L) chains of MEDI-528.

In certain embodiments, the present invention provides a compositioncomprising a fusion protein. In some embodiments, the fusion proteincomprises an Fc domain of an antibody or a fragment thereof, wherein theFc domain of Fc domain fragment comprises at least one thioethercross-link. In other embodiments, the fusion protein comprises anC_(H)1, C_(H)2, C_(H)3 and/or C_(L) domain of an antibody, wherein theC_(H)1, C_(H)2, C_(H)3 or C_(L) domain comprises at least one thioethercross-link. In another embodiments, the fusion protein comprises two,three or all of the domains of C_(H)1, C_(H)2, C_(H)3 or C_(L).

In a further aspect, the present invention provides a compositionsubstantially free of a denaturing reagent comprising a polypeptide,wherein the polypeptide comprises a lanthionine. A lanthionine residueis known to those skilled in the art, as a sulfur bridged alanine dimer,having the structure of (—OOC—CH(NH—)—CH₂—S—CH₂—CH(NH—)—COO—). In someembodiments, the polypeptide is more than 2%, 5%, 10% or 15% of thepolypeptide molecules of the composition. In certain embodiments, thepolypeptide is an antibody. The antibodies in the composition may be,but are not limited to polyclonal antibodies, monoclonal antibodies, Fabfragments, F(ab)′ fragments, F(ab)′₂ fragments, single chain antibodies,human antibodies, humanized or chimeric antibodies, and epitope-bindingfragments of any of the above. The lanthionine can be located in anyposition of a polypeptide. For example, the thioether cross-link can beat the N-terminus, at the C-terminus, or between the N-terminus andC-terminus of a polypeptide chain.

In a further aspect, the present invention provides a compositionsubstantially free of a denaturing reagent comprising a population ofantibodies, i.e., two, three, four, five or more antibodies, wherein atleast 2%, 5%, 10%, 15% or 20% of the antibodies comprise at least onethioether cross-link. In some embodiments, about 2-20%, 2-15%, 2-10%,2-5%, 5-20%, 10-15%, 10-30%, 20-30%, 10-40%, 10-50%, 20-50% or 10-75% ofthe antibodies comprise at least one thioether cross-link.

In certain embodiments, at least 2%, 5%, 10%, 15% or 20% of theantibodies comprise at least two thioether cross-link. In someembodiments, about 2-20%, 2-15%, 2-10%, 2-5%, 5-20%, or 10-15% of theantibodies comprise at least two thioether cross-link.

In certain embodiments, the composition is a pharmaceutical composition.In some embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carriers.

In certain embodiments, populations of antibodies all have the sameamino acid sequence of V_(H) and V_(L) chains. In some embodiments,populations of antibodies all have the same CDRs of V_(H) and V_(L)chains.

In certain embodiments, populations of antibodies have different aminoacid sequence of V_(H) and V_(L) chains. In some embodiments,populations of antibodies have different CDRs of V_(H) and V_(L) chains.

The invention provides a composition substantially free of a denaturingreagent comprising a population of fusion proteins, i.e. two, three,four, five or more fusion proteins, wherein at least 2%, 5%, 10%, 15% or20% of the fusion proteins comprise an Fc domain or a fragment thereofcomprising at least thioether cross-link. In some embodiments, about2-20%, 2-15%, 2-10%, 2-5%, 5-20%, 10-15%, 10-30%, 20-30%, 10-40%,10-50%, 20-50% or 10-75% of the antibodies comprise at least onethioether cross-link. In certain embodiments, the population of fusionproteins have the same amino acid sequence. In other embodiments, thepopulation of fusion proteins have different amino acid sequences. In aspecific embodiment, the composition is a pharmaceutical composition.

The present invention provides a composition comprising a population ofantibodies, i.e., two, three, four, five or more antibodies, whereinless than 2%, 1.5%, 1% or 0.5% of the antibodies in the populationcomprise a thioether cross-link. In certain embodiments, the populationof the antibodies have the same amino acid sequences. In otherembodiments, the population of antibodies have different amino acidsequences. In a specific embodiment, the composition is a pharmaceuticalcomposition.

The present invention provides a composition a population of fusionproteins, i.e., two, three, four, five or more fusion proteins, whereinless than 2%, 1.5%, 1% or 0.5% of the fusion proteins in the populationcomprise a thioether cross-link. In certain embodiments, the populationof antibodies have the same amino acid sequences. In other embodiments,the population of fusion proteins have different amino acid sequences.In a specific embodiment, the composition is a pharmaceuticalcomposition.

In another aspect, the present invention provides an isolated antibody,wherein the antibody comprises at least one thioether cross-link, thatbinds to one or more particular antigens. In certain embodiments, theantibody of the present invention specifically binds to an antigen ofrespiratory syncytial virus (RSV). In some embodiments, the antibodycomprises the amino acid sequence of the variable heavy (V_(H)) andvariable light (V_(L)) chains of palivizumab or motavizumab. In otherembodiments, the antibody comprises the amino acid sequence of thecomplementarity determining regions (CDRs) of the V_(H) and V_(L) chainsof palivizumab or motavizumab.

In certain embodiments, the antibody of the present inventionspecifically binds to an antigen of human metapneumovirus (hMPV). Insome embodiments, the antibody is a humanized antibody that specificallybinds to an antigen of hMPV.

In certain embodiments, the antibody of the present inventionspecifically binds to integrin α_(v)β₃. In some embodiments, theantibody comprises the amino acid sequence of the V_(H) and V_(L) chainsof MEDI-522 (Vitaxin®). In other embodiments, the antibody comprises theamino acid sequence of the CDRs of the V_(H) and V_(L) chains ofMEDI-522 (Vitaxin®).

In certain embodiments, the antibody of the present inventionspecifically binds to CD2. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of siplizumab. Inother embodiments, the antibody comprises the amino acid sequence of thecomplementarity determining regions (CDRs) of V_(H) and V_(L) chains ofsiplizumab.

In certain embodiments, the antibody of the present inventionspecifically binds to CD19. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of MT103. In otherembodiments, the antibody comprises the amino acid sequence of the CDRsof the V_(H) and V_(L) chains of MT103.

In certain embodiments, the antibody of the present inventionspecifically binds to an Eph receptor. In certain embodiments, theantibody of the present invention specifically binds to EphA2. In someembodiments, the antibody comprises the amino acid sequence of the V_(H)and V_(L) chains of EA2 or EA5. In other embodiments, the antibodycomprises the amino acid sequence of the CDRs of the V_(H) and V_(L)chains of EA2 or EA5. In certain embodiments, the antibody of thepresent invention specifically binds to EphA4. In some embodiments, theantibody of the present invention specifically binds to EphB4.

In certain embodiments, the antibody of the present inventionspecifically binds to IL-9. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of MEDI-528. Inother embodiments, the antibody comprises the amino acid sequence of theCDRs of the V_(H) and V_(L) chains of MEDI-528.

In another aspect, the present invention provides a fusion proteincomprising at least one thioether cross-link. In some embodiments, thefusion protein comprises an Fc domain of an antibody or a fragmentthereof, wherein the Fc domain or Fc domain fragment comprises at leastone thioether cross-link. In other embodiments, the fusion proteincomprises an C_(H)1, C_(H)2, C_(H)3 and/or C_(L) domain of an antibody,wherein the C_(H)1, C_(H)2, C_(H)3 or C_(L) domain comprises at leastone thioether cross-link. In another embodiments, the fusion proteincomprises two, three or all of the domains Of C_(H)1, C_(H)2, C_(H)3 orC_(L).

In another aspect, the present invention provides a method forincreasing the amount of an antibody in a composition, wherein theantibody comprises at least one thioether cross-link. The antibody inthe composition can be any antibody comprising at least one thioethercross-link as described herein. In certain embodiments, the methodcomprises incubating the composition at a temperature greater than 4°C., or at pH greater than 7, for a time sufficient to increase theamount of said antibody species. In some embodiments, the method furthercomprises contacting the composition with a reducing agent.

In a further aspect, the present invention provides a method forproducing a composition enriched in an antibody, wherein the antibodycomprises at least one thioether cross-link. The antibody in thecomposition can be any antibody comprising at least one thioethercross-link as described herein. In certain embodiments, the method ofthe invention comprises incubating the composition at a temperaturegreater than 4° C., or at pH greater than 7, for a time sufficient toenrich said antibody species. In some embodiments, the method furthercomprises contacting the composition with a reducing agent.

In another aspect, the present invention provides a method fordecreasing the amount of an antibody in a composition, wherein saidantibody comprises at least one thioether cross-link in a compositionresulting from a purification method. In certain embodiments, the methodcomprises reducing in pH and/or temperature of at least one step of saidpurification method resulting in a lower level of said antibodycomprising at least one thioether cross-link than said firstpurification method.

In a further aspect, the present invention provides a method forproducing a composition enriched in a fusion, wherein the fusion proteincomprises at least one thioether cross-link. The fusion protein in thecomposition can be any fusion protein comprising at least one thioethercross-link as described herein. In certain embodiments, the method ofthe invention comprises incubating the composition at a temperaturegreater than 4° C., or at pH greater than 7, for a time sufficient toenrich said fusion protein species. In some embodiments, the methodfurther comprises contacting the composition with a reducing agent.

In another aspect, the present invention provides a method fordecreasing the amount of a fusion protein in a composition, wherein saidfusion protein comprises at least one thioether cross-link in acomposition resulting from a purification method. In certainembodiments, the method comprises reducing in pH and/or temperature ofat least one step of said purification method resulting in a lower levelof said fusion protein comprising at least one thioether cross-link thansaid first purification method.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show reducing CGE (panel A) and SDS-PAGE (panel B) analysisof a IgG1 monoclonal antibody. Lane 1 is molecular weight markers; Lane2 and Lane 3 are a reduced monoclonal antibody. Panel C shows thegraphical view of the heavy chain, light chain and 92 kDa cross-linkedspecies.

FIG. 2 shows size exclusion chromatogram of a reduced and alkylatedmonoclonal antibody motavizumab.

FIGS. 3A and 3B show tryptic peptide map profiles of the early-elutingSEC fraction (panel A) and unfractionated monoclonal antibody (panel B).

FIGS. 4A and 4B show MS/MS spectra and assignment of fragment ions ofthe cross-linked peptide of L19-L20 (SFNRGEC) and H19 (SCDK) with athioether bond linkage (ion at m/z 615.6^(±2)) (panel A) and a disulfidebond linkage (ion at m/z 631.6^(±2)) (panel B). Note: “e” in the figurerefers to the side chain cleavage on the Cys and not the amino acidbackbone. “2” refers to the Cys in the CSDK peptide chain. “7” refers tothe Cys in the SFRNGEC peptide chain.

FIGS. 5A and 5B show deconvoluted mass spectra of the Fab fragment of amonoclonal antibody stored at 4° C. (panel A) and at 40° C. (panel B).

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery andcharacterization of a non-reducible thioether bridge between the heavyand light chains of different monoclonal antibodies. Accordingly, theinvention provides macromolecules comprising a thioether cross-link,wherein the thioether cross-link is a thioether comprising a singlethioether bond linking two residues of a macromolecule.

5.1. DEFINITIONS

As used herein, the term “isolated” in the context of a proteinaceousagent (e.g., peptide, polypeptide, fusion protein or antibody) refers toa proteinaceous agent which is substantially free of cellular materialor contaminating proteins from the cell or tissue source from which itis derived, or substantially free of chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of a proteinaceous agent inwhich the proteinaceous agent is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus, aproteinaceous agent that is substantially free of cellular materialincludes preparations of a proteinaceous agent having less than about30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (alsoreferred to herein as a “contaminating protein”). When the proteinaceousagent is recombinantly produced, it is also preferably substantiallyfree of culture medium, i.e., culture medium represents less than about20%, 10%, or 5% of the volume of the protein preparation. When theproteinaceous agent is produced by chemical synthesis, it is preferablysubstantially free of chemical precursors or other chemicals, i.e., itis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the proteinaceous agent. Accordingly suchpreparations of a proteinaceous agent have less than about 30%, 20%,10%, 5% (by dry weight) of chemical precursors or compounds other thanthe proteinaceous agent of interest.

As used herein, the term “humanized antibody” refers to forms ofnon-human (e.g., murine) antibodies that are chimeric antibodies whichcontain minimal sequence derived from non-human immunoglobulin. For themost part, humanized antibodies are human immunoglobulins (recipientantibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-human species(donor antibody) such as mouse, rat, rabbit or non-human primate havingthe desired specificity, affinity, and capacity. In some instances,Framework Region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiesmay comprise residues which are not found in the recipient antibody orin the donor antibody. These modifications are made to further refineantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin that immunospecifically binds to an EphA2polypeptide, that has been altered by the introduction of amino acidresidue substitutions, deletions or additions (i.e., mutations). In someembodiments, a humanized antibody is a derivative. Such a humanizedantibody comprises amino acid residue substitutions, deletions oradditions in one or more non-human CDRs. The humanized antibodyderivative may have substantially the same binding, better binding, orworse binding when compared to a non-derivative humanized antibody. Inspecific embodiments, one, two, three, four, or five amino acid residuesof the CDR have been substituted, deleted or added (i.e., mutated). Forfurther details in humanizing antibodies, see European Patent Nos. EP239,400, EP 592,106, and EP 519,596; International Publication Nos. WO91/09967 and WO 93/17105; U.S. Pat. Nos. 5,225,539, 5,530,101,5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991,Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, ProteinEngineering 7(6):805-814; Roguska et al., 1994, PNAS 91:969-973; Tan etal., 2002, J. Immunol. 169:1119-25; Caldas et al., 2000, Protein Eng.13:353-60; Morea et al., 2000, Methods 20:267-79; Baca et al., 1997, J.Biol. Chem. 272:10678-84; Roguska et al., 1996, Protein Eng. 9:895-904;Couto et al., 1995, Cancer Res. 55 (23 Supp):5973s-5977s; Couto et al.,1995, Cancer Res. 55:1717-22; Sandhu, 1994, Gene 150:409-10; Pedersen etal., 1994, J. Mol. Biol. 235:959-73; Jones et al., 1986, Nature321:522-525; Reichmann et al., 1988, Nature 332:323-329; and Presta,1992, Curr. Op. Struct. Biol. 2:593-596, and Wu et al., U.S. Pat.Application Publication. No. 2005/0042664, published Feb. 24, 2004.

As used herein, the terms “single-chain Fv” or “scFv” refer to antibodyfragments comprise the VH and VL domains of antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. For a review of sFv see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994). In specific embodiments,scFvs include bispecific scFvs and humanized scFvs.

As used herein, the term “antigen” refers to a macromolecule that isrecognized by antibodies and can trigger an immune response. An antigencan be a protein or a polysaccharide, but it can also be any type ofmolecule, even small molecules if coupled to a larger carrier.

As used herein, the term “epitopes” refers to fragments of a polypeptideor protein having antigenic or immunogenic activity in an animal,preferably in a mammal, and most preferably in a human. An epitopehaving immunogenic activity is a fragment of a polypeptide or proteinthat elicits an antibody response in an animal. An epitope havingantigenic activity is a fragment of a polypeptide or protein to which anantibody immunospecifically binds as determined by any method well-knownto one of skill in the art, for example by immunoassays. Antigenicepitopes need not necessarily be immunogenic.

As used herein, the terms “protein” and the term “polypeptide,” usedinterchangeably, to refer to a complex organic compound composed ofamino acid residues joined by peptide bonds.

As used herein, the term “specifically binds to an antigen” andanalogous terms refer to peptides and polypeptides (e.g., fusionproteins and antibodies) that specifically bind to an antigen or afragment thereof and do not specifically bind to other antigens. Apeptide or polypeptide (e.g., fusion proteins and antibodies) thatspecifically binds to an antigen may bind to other peptides orpolypeptides with lower affinity as determined by, e.g., immunoassays,BIAcore, or other assays known in the art. Peptides and polypeptides(e.g., fusion proteins and antibodies) that specifically bind to anantigen may cross-reactive with related antigens. Preferably, antibodiesthat specifically bind to an antigen do not cross-react with otherantigens.

When describing the macromolecules, compositions containing suchmacromolecules and methods of using such macromolecules andcompositions, the following terms have the following meanings unlessotherwise indicated.

“Acyl” refers to a radical —C(O)R, where R is hydrogen, alkyl,cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl,heteroarylalkyl as defined herein. Representative examples include, butare not limited to, formyl, acetyl, cylcohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.

“Aliphatic” refers to hydrocarbyl organic compounds or groupscharacterized by a straight, branched or cyclic arrangement of theconstituent carbon atoms and an absence of aromatic unsaturation.Aliphatics include, without limitation, alkyl, alkylene, alkenyl,alkenylene, alkynyl and alkynylene. Aliphatic groups typically have from1 or 2 to about 12 carbon atoms.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 11 carbon atoms, more particularly as alower alkyl, from 1 to 8 carbon atoms and still more particularly, from1 to 6 carbon atoms. The hydrocarbon chain may be eitherstraight-chained or branched. This term is exemplified by groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl,n-hexyl, ii-octyl, tert-octyl and the like. The term “lower alkyl”refers to alkyl groups having 1 to 6 carbon atoms. The term “alkyl” alsoincludes “cycloalkyl” as defined below.

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 11 carbon atoms and more particularly 1to 6 carbon atoms which can be straight-chained or branched. This termis exemplified by groups such as methylene (—CH2-), ethylene (—CH2CH2-),the propylene isomers (e.g., —CH2CH2CH2- and —CH(CH3)CH2-) and the like.

“Aryl” refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene and the like. Particularly, anaryl group comprises from 6 to 14 carbon atoms.

“Aralkyl” or “arylalkyl” refers to an alkyl group, as defined above,substituted with one or more aryl groups, as defined above.

“Amino” refers to the radical —NH₂.

“Aminocarbonyl” or “amido” refers to the group —C(O)NRR where each R isindependently hydrogen, alkyl, aryl and cycloalkyl, or where the Rgroups are joined to form an alkylene group.

“Carbamoyl” refers to the radical —C(O)N(R)₂ where each R group isindependently hydrogen, alkyl, cycloalkyl or aryl, as defined herein,which may be optionally substituted as defined herein.

“Cycloalkyl” refers to cyclic hydrocarbyl groups having from 3 to about10 carbon atoms and having a single cyclic ring or multiple condensedrings, including fused and bridged ring systems, which optionally can besubstituted with from 1 to 3 alkyl groups. Such cycloalkyl groupsinclude, by way of example, single ring structures such as cyclopropyl,cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl,2-methylcyclopentyl, 2-methylcyclooctyl, and the like, and multiple ringstructures such as adamantanyl, and the like.

“Hetero” when used to describe a compound or a group present on acompound means that one or more carbon atoms in the compound or grouphave been replaced by a nitrogen, oxygen, or sulfur heteroatom. Heteromay be applied to any of the hydrocarbyl groups described above such asalkyl, e.g. heteroalkyl, cycloalkyl, e.g. cycloheteroalkyl, aryl, e.g.heteroaryl, cycloalkenyl, cycloheteroalkenyl, and the like having from 1to 5, and especially from 1 to 3 heteroatoms.

“Heteroaryl” or “heteroaromatic” refers to a monovalent heteroaromaticgroup derived by the removal of one hydrogen atom from a single atom ofa parent heteroaromatic ring system. Typical heteroaryl groups include,but are not limited to, groups derived from acridine, arsindole,carbazole, -carboline, chromane, chromene, cinnoline, furan, imidazole,indazole, indole, indoline, indolizine, isobenzofuran, isochromene,isoindole, isoindoline, isoquinoline, tetrahydroisoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, tetrahydroquinoline,quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene,triazole, xanthene, and the like. Particularly, heteroaryl can includeother saturated ring systems, and can therefore be derived fromindoline, indolizine, tetrahydroquinoline, and tetrahydroisoquinoline.Preferably, the heteroaryl group is between 5-20 membered heteroaryl,with 5-10 membered heteroaryl being particularly preferred. Particularheteroaryl groups are those derived from thiophene, pyrrole,benzothiophene, benzofuran, indole, pyridine, pyrimidine, quinoline,tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline, imidazole,oxazole and pyrazine.

“Hetero substituent” refers to a halo, O, S or N atom-containingfunctionality that may be present as an R4 in a CR4 group present assubstituents directly on W or Z of the compounds of this invention ormay be present as a substituent in the “substituted” aryl, heteroaryland aliphatic groups present in the compounds.

Examples of hetero substituents include:

-halo,

—NO₂, —NH₂, —NHR, —N(R)₂,

—NRCOR, —NRSOR, —NRSO₂R, OH, CN, CO₂R,

—COOH,

—O—R,

—CON(R)₂, —CONROR,

—SO₃H, —S—R, —SO₂N(R)₂,

—S(O)R, and —S(O)₂R,

wherein each R is independently an aryl or aliphatic, optionally withsubstitution. Among hetero substituents containing R groups, preferenceis given to those materials having aryl and alkyl R groups as definedherein. Where feasible, each R may include hydrogen. Also, wherefeasible, two R groups when on same atom may join to form a heterocyclicring of 3-8 atoms. For example, two R groups of NR2, SO2NR2, and CONR2may join, together with the N atom, to form a N-morpholino, N-pyrrolo,N-piperidino, and N-pyrazolylo ring. Preferred hetero substituents arethose listed above.

As used herein, the term “thioether cross-link” refers to a singlethioether bond between two residues of a macromolecule. For purpose ofthis invention, the two residues of the macromolecules are linked by asingle sulfur atom. Links that comprise two or more sulfur atoms, forinstance, disulfide bridges, are not thioether cross-links of thepresent invention. A preferred thioether cross-link can be found in alanthionine residue, where a single sulfur atom bridges the side chainsof two amino acids. Other links having a single sulfur atom aredescribed herein in detail. As will be recognized by those skilled inthe art, the formation of a thioether cross-link can result in the lossof one or more atoms, such as a sulfur or hydrogen atoms, from one orboth of the residues. Certain thioether cross-links have a structureaccording to formula I: —OOC—CH(NH—)—R¹—S—R²—CH(NH—)—COO—, wherein R¹and R² are independently side chains of macromolecule residues, such asamino acid residues. The residue can be natural or non-natural. Incertain embodiments, R¹ and R² can lose one or more atoms, for instance,sulfur or hydrogen atoms in the formation of the thioether cross-link.Preferred R¹ and R² groups include methylene, ethylene, propylene, andbutylene. Particularly preferred is methylene. The term “thioethercross-link” is intended to be interchangeable with the term “zero-orderthioether” of provisional application No. 60/661,724, the contents ofwhich are incorporated by reference in its entirety.

As used herein, a compositions that is “substantially free of adenaturing reagent” refers to a composition free of denaturing reagentor having only denaturing reagents in such an amount that macromoleculesin the compositions maintain their three-dimensional structures. Incertain embodiments, at least 50% of the macromolecules in thecomposition are in their native states.

As used herein, the term “disease” or “disorder,” used interchangeably,refers to a condition, e.g., a pathogenic condition, in a subject.

As used herein, the terms “therapies” and “therapy” (e.g., aprophylactic agent or a therapeutic agent) can refer to any protocol(s),method(s) and/or agent(s) that can be used in the prevention, treatment,management or amelioration of a disorder or one or more symptomsthereof.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of a disorder,or prevention of recurrence or spread of a disorder.

As used herein, the terms “prophylactically effective amount” may referto the amount of prophylactic agent sufficient to prevent the recurrenceor spread of a disease or the occurrence of such in a patient, includingbut not limited to those predisposed to the disease. A prophylacticallyeffective amount may also refer to the amount of the prophylactic agentthat provides a prophylactic benefit in the prevention of disease.Further, a prophylactically effective amount with respect to aprophylactic agent of the invention means that amount of prophylacticagent alone, or in combination with other agents, that provides aprophylactic benefit in the prevention of disease.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment, management oramelioration a disorder or one or more symptoms thereof.

As used herein, a “therapeutically effective amount” refers to thatamount of the therapeutic agent sufficient to treat or manage a disorderor a symptom thereof. A therapeutically effective amount may refer tothe amount of a therapy sufficient to delay or minimize the onset ofdisorder, e.g., delay or minimize the spread of cancer. Atherapeutically effective amount may also refer to the amount of thetherapy that provides a therapeutic benefit in the treatment ormanagement of a disorder. A therapeutically effective amount may alsorefer to the amount of the therapy that reduces the progression,severity and/or duration in the treatment or management of a disorder.

As used herein, the terms “prevent”, “preventing” and “prevention” inthe context of the administration of a therapy, refer to the preventionof the recurrence or onset of a disorder or one or more symptoms thereofin a subject as result of the administration of a prophylactic ortherapeutic agent.

As used herein, the terms “treat,” “treating” and “treatment” in thecontext of the administration of a therapy refer to the reduction oramelioration of the progression, severity, and/or duration of a disorderor the eradication, reduction or amelioration of symptoms of a disorder,(e.g., the eradication, removal, modification, or control of primary,regional, or metastatic cancer tissue) that results from theadministration of one or more therapies. In certain embodiments, suchterms refer to the minimizing or delaying the spread of cancer resultingfrom the administration of one or more therapeutic agents to a subjectwith such a disease.

As used herein, the terms “manage”, “managing” and “management” in thecontext of the administration of a therapy, refer to the beneficialeffects that a subject derives from a therapy, which does not result ina cure of the disorder. In certain embodiments, a subject isadministered one or more therapy to “manage” a disorder so as to preventthe progression or worsening of the disorder (i.e., hold diseaseprogress).

As used herein, the term “in combination” refers to the use of one ormore therapies. The use of the term “in combination” does not restrictthe order in which prophylactic and/or therapeutic agents areadministered to a subject with a disorder. A first therapies can beadministered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapies to a subjectwith a disorder.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is preferably a mammal suchas a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and aprimate (e.g., monkey and human), most preferably a human.

5.2. MACROMOLECULES COMPRISING THIOETHER CROSS-LINKS

As discussed in detail in the section below, the present inventionprovides macromolecules that comprise a thioether cross-link, andcompositions comprising the macromolecules. The prevent invention alsoprovides methods of using the macromolecules. Further, the presentinvention provides methods of producing and using the compositionscomprising the macromolecules.

The macromolecules of the present invention can be any type ofmacromolecule as recognized by those skilled in the art. In someembodiments, the macromolecules of the present invention arepolypeptides. The macromolecules of the present invention can be anytype of polypeptides recognized by those skilled in the art, such asenzymes, antibodies, membrane-associated proteins, or secreted proteins,etc. In certain embodiments, the present invention encompassescytokines, including but not limited to interleukins and interferons. Inanother embodiments, the present invention encompassesmembrane-associated proteins, for instance, receptors or ion channels.In yet another embodiments, the present invention provides antibodies,wherein said antibodies comprises a thioether cross-link.

A thioether cross-link is any thioether bond that meets a definitionprovided herein. As discussed above, a thioether cross-link is a linkbetween residues of a polypeptide, wherein the link has a single sulfuratom. Significantly, thioether cross-links expressly do not includelinks that comprise more than one sulfur atom, such as disulfide bridgesthat are familiar to those of skill in the art. Instead, a thioethercross-link has a single sulfur atom that bridges residues of amacromolecule.

The atoms that bridge residues of a macromolecule are those atoms thatlink one residue to another residue as recognized by those of skill inthe art. In preferred embodiments, these atoms consist of the smallestnumber of atoms that connect a portion of one residue of themacromolecule to other residues of the macromolecule. In preferredembodiments, the atoms that bind residues of a macromolecule are notbackbone atoms, as understood by those skilled in the art. These atomsform a thioether cross-link when they comprise no more than a singlesulfur atom.

For instance, in certain embodiments, the thioether cross-link links theside chains of residues of the macromolecules. The atoms that link oneside chain to the other side chains should comprise no more than asingle sulfur atom. When the linked side chains comprise a single sulfuratom, the side chains are linked by a thioether cross-link. In certainembodiments, the residues may comprise other sulfur atoms so long as theother sulfur atoms are not necessary to bridge the residues. In furtherembodiments, the residues comprise only a single sulfur atom.

The residues linked by the thioether cross-link can be natural residuesor non-natural residues. Formation of the thioether cross-link canresult in the loss of atoms from the residues, as will be recognized bythose of skill in the art. For instance, formation of a thioethercross-link between side chains of two cysteine residues can result inthe loss of a sulfur atom and hydrogen atoms from the residues, yet theresulting thioether cross-link will be recognized as linking thecysteine residues by one of skill in the art.

The thioether cross-link can link any two residues of a polypeptide. Inpreferred embodiments, the residues linked by the thioether cross-linkare natural residues. In particular embodiments, one or both of theresidues are selected from the group consisting of cysteine, asparticacid, glutamic acid, histidine, methionine and tyrosine. In furtherembodiments, two of the residues are selected from the group consistingof cysteine, aspartic acid, glutamic acid, histidine, methionine andtyrosine. In preferred embodiments, two of the residues are cysteineresidues.

The macromolecule of the present invention can comprise one or morethioether cross-links. In some embodiments, the macromolecule comprisesonly one thioether cross-link. In other embodiments, the macromoleculecomprises two, three or more thioether cross-links.

In certain embodiments the thioether cross-link has the structure offormula (I): —OOC—CH(NH—)—R¹—S—R²—CH(NH—)—COO—, or any salt or solvatethereof. In formula I, the dashes indicate bonds. The dashes from thecarboxy and amino groups can be bonds to other portions of themacromolecule or to hydrogen or to groups known to those of skill in theart to modify amino or carboxy termini of macromolecules. R¹ and R² areeach independently side chains of natural or non-natural amino acidsthat would result from the formation of the thioether cross-link offormula I. In certain embodiments, each of R¹ and R² is independentlyselected from the group consisting of a bond, alkyl, heteroalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, alkylacyl, alkylamino andalkylacylamino. The sulfur can be bonded to any atom of R¹ and R² wherechemically feasible as will be recognized by those of skill in the art.In preferred embodiments, each of R¹ and R² is independently C₁-C₇alkyl, C₁-C₆ alkyl, C₁-C₅ alkyl, C₁-C₄ alkyl, C₁-C₃ alkyl, C₁-C₂ alkylor C₁ alkyl. In particularly preferred embodiments, each of R¹ and R² isindependently methylene.

The thioether cross-link can be at any location of the macromoleculewhere feasible according to the knowledge of those of skill in the art.For instance, the thioether cross-link can be inter-molecular orintra-molecular. In some embodiments, the thioether cross-link isinter-molecular. For example, the thioether cross-link can link twopolypeptide chains of an antibody. In some embodiments, the thioethercross-link links a heavy chain and a light chain of the antibody. Inother embodiments, the thioether cross-link links two heavy chains ortwo light chains of the antibody.

Within a polypeptide chain, a residue of the thioether cross-link can belocated in any position apparent to those of skill in the art. In someembodiments, a residue of the thioether cross-link is located at theN-terminus of a polypeptide. In other embodiments, a residue of thethioether cross-link is located at the C-terminus of a polypeptide. Infurther embodiments, a residue the thioether cross-link is locatedbetween the N-terminus and C-terminus of a polypeptide. In someembodiments, the thioether cross-link can link a residue in the middleof a polypeptide to a residue at the N or C-terminus of a polypeptide.In a particular embodiment, the thioether cross-link can link theresidue at the N-terminus of a polypeptide, to the residue at theC-terminus of the same polypeptide thereby forming a cyclic polypeptide.

In certain antibody embodiments, the thioether links a cysteine residueof a heavy chain and a cysteine residue of a light chain. The cysteineresidues can be in any region of the heavy chain or light chain. Incertain embodiments, the cysteine residue of the heavy chain is in theC_(H)1 region and the cysteine residue of the light chain is in C_(L)region. In one embodiment, the cysteine residue in the CHI region of theheavy chain is at the amino acid position 223, and the cysteine residuein the C_(L) region of the heavy chain is at the amino acid position213.

In certain embodiments, the present invention provides a macromoleculecomprising lanthionine or a composition comprising a macromolecule,wherein the macromolecule comprises lanthionine. As will be recognizedby those of skill in the art, lanthionine is a sulfide bridged alaninedimer (—OOC—CH(NH—)—CH₂—S—CH₂—CH(NH—)—COO—). Lanthionine, andmacromolecules comprising lanthionine, can be prepared by methodsdescribed herein or by methods apparent to those of skill in the art.For instance, lanthionine can be prepared from cysteine anddehydrolalanine, or from cysteine and cysteine, by post-translationalmodification of peptides. In certain embodiments, the macromolecule is apolypeptide, In further embodiments, the polypeptide is an antibody.

In some embodiments, polypeptides, preferably, antibodies, can bemodified to incorporate a thioether cross-link according to theinvention. For example, any residue can be substituted with a cysteineby techniques known to those of skill in the art. For instance, standardtechniques can be used to introduce mutations in the nucleotide sequenceencoding an antibody, or fragment thereof, including, e.g.,site-directed mutagenesis and PCR-mediated mutagenesis, which results inamino acid substitutions. In a preferred embodiments, one or morenon-essential amino acid residues of the polypeptide can be substitutedwith a cysteine. In certain embodiments, the antibody one or morenon-essential amino acid residues substituted with a cysteine. Incertain embodiments, the substitutions with a cysteine are conservativeamino acid substitution made at one or more predicted non-essentialamino acid residues (i.e., amino acid residues which are not criticalfor the antibody to immunospecifically bind to an antigen). A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

5.3. COMPOSITIONS COMPRISING MACROMOLECULES THAT COMPRISE THIOETHERCROSS-LINK

In one aspect, the prevent invention provides compositions substantiallyfree of a denaturing reagent comprising a macromolecule, wherein saidmacromolecule comprises at least one thioether cross-link. In certainembodiments, the macromolecule comprises two, three, four, five or morethioether cross-links. In other embodiments, the macromolecules comprise1-3, 1-5, 2-5, 2-4, 1-10, 5-10, or 2-6 thioether cross-links. In someembodiments, the macromolecules are polypeptides. In some furtherembodiments, the macromolecules are antibodies. The antibodiescomprising at least one thioether cross-link are described in detail inSection 5.2 and 5.4.

A composition that is “substantially free of a denaturing reagent”refers to a composition free of any denaturing reagent or having onlydenaturing reagents in such an amount that macromolecules in thecompositions maintain their three-dimensional structures and thus theircharacteristic folded structures. In some embodiments, at least 50% ofthe macromolecules in the composition are in their native states. Insome embodiments, at least 60% of the macromolecules in the compositionare in their native states. In some embodiments, at least 70% of themacromolecules in the composition are in their native states. In someembodiments, at least 80% of the macromolecules in the composition arein their native states. In some embodiments, at least 90% of themacromolecules in the composition are in their native states. In someembodiments, at least 95% of the macromolecules in the composition arein their native states. In some embodiments, at least 99% of themacromolecules in the composition are in their native states. In someembodiments, about 50%-99.99%, 50%-99%, 50%-95%, 50%-90%, 50%-80%,50%-70%, 50%-60%, 60%-99%, 70%-99%, 80%-99%, 90%-99%, 60%-95%, or70%-90% of the macromolecules in the composition are in their nativestates.

A denaturing reagent is any reagent that might cause a structure changein an macromolecule, including but limited to acids, bases ordetergents. Exemplary denaturing reagents include, for example, sodiumdodecyl sulfate, urea, and guanidine. The amount of denaturing reagentsin the composition to be qualified as “substantially free of adenaturing reagent” depends on the nature of the denaturing reagents andthe particular antibodies in the composition. The stronger thedenaturing reagents, the less the denaturing reagent should be in thecomposition. In some embodiments, the denaturing reagents are less than50% of the composition. In some embodiments, the denaturing reagents areless than 25% of the composition. In some embodiments, the denaturingreagents are less than 10% of the composition. In some embodiments, thedenaturing reagents are less than 5% of the composition. In someembodiments, the denaturing reagents are less than 2.5% of thecomposition. In some embodiments, the denaturing reagents are less than1% of the composition. In some embodiments, the denaturing reagents areless than 0.5% of the composition. In some embodiments, the denaturingreagents are less than 0.25% of the composition. In some embodiments,the denaturing reagents are less than 0.1% of the composition. In someembodiments, the denaturing reagents are about 0.1%-50%, 0.1%-25%,0.1%-10%, 0.1%-5%, 0.1%-2.5%, 0.1%-1%, 0.1%-0.5%, 0.1%-0.25%, 0.25%-50%,0.25%-25%, 0.5%-10%, or 0.5%-1% of the composition.

In some embodiments, the macromolecules comprising at least onethioether cross-link are more than 2%, 5%, 10%, 15%, 25%, 35%, 45%, 50%or 75% of the total macromolecules in the composition. In certainembodiments, the macromolecules comprising at least one thioethercross-link are more than 2% of the total macromolecules in thecomposition. In certain embodiments, the macromolecules comprising atleast one thioether cross-link are more than 5% of the totalmacromolecules in the composition. In certain embodiments, themacromolecules comprising at least one thioether cross-link are morethan 10% of the total macromolecules in the composition. In certainembodiments, the macromolecules comprising at least one thioethercross-link are more than 15% of the total macromolecules in thecomposition. In some embodiments, the macromolecules comprising at leastone thioether cross-link are about 2%-15%, 2%-10%, 2%-5%, 5%-15%,5%-10%, 10%-15%, 10-30%, 20-30%, 10-40%, 10-50%, 20-50% or 10-75% of thetotal macromolecules in the composition.

In other embodiments, the macromolecules comprising at least onethioether cross-link are less than 4%, 2%, 1%, 0.75%, 0.5%, 0.25%, 0.1%and 0.05% of the total macromolecules in the composition. In certainembodiments, the macromolecules comprising at least one thioethercross-link are less than 4% of the total macromolecules in thecomposition. In certain embodiments, the macromolecules comprising atleast one thioether cross-link are less than 2% of the totalmacromolecules in the composition. In certain embodiments, themacromolecules comprising at least one thioether cross-link are lessthan 1% of the total macromolecules in the composition. In certainembodiments, the macromolecules comprising at least one thioethercross-link are less than 0.75% of the total macromolecules in thecomposition. In certain embodiments, the macromolecules comprising atleast one thioether cross-link are less than 0.5% of the totalmacromolecules in the composition. In certain embodiments, themacromolecules comprising at least one thioether cross-link are lessthan 0.25% of the total macromolecules in the composition. In certainembodiments, the macromolecules comprising at least one thioethercross-link are less than 0.1% of the total macromolecules in thecomposition. In certain embodiments, the macromolecules comprising atleast one thioether cross-link are less than 0.05% of the totalmacromolecules in the composition. In some embodiments, themacromolecules comprising at least one thioether cross-link are about0.05%-4%, 0.05%-2%, 0.05%-1%, 0.05%-0.5%, 0.05%-0.25%, 0.05%-0.1%,0.1%-4%, 0.25%-2%, 0.5%-1% of the total macromolecules in thecomposition.

In a further aspect, the present invention provides compositionssubstantially free of a denaturing reagent comprising an antibody,wherein the antibody comprises at least one thioether cross-link andwherein the antibody specifically binds to one or more particularantigens. In another aspect, the present invention provides an isolatedantibody that comprises at least one thioether cross-link, wherein theantibody specifically binds to one or more particular antigens.

In certain embodiments, the antibody of the present inventionspecifically binds to an antigen of respiratory syncytial virus (RSV).In some embodiments, the antibody comprises the amino acid sequence ofthe variable heavy (V_(H)) and variable light (VL) chains of palivizumabor motavizumab. In other embodiments, the antibody comprises the aminoacid sequence of the complementarity determining regions (CDRs) of theV_(H) and V_(L) chains of palivizumab or motavizumab.

In certain embodiments, the antibody of the present inventionspecifically binds to an antigen of human metapneumovirus (hMPV). Insome embodiments, the antibody is a humanized antibody that specificallybinds to an antigen of hMPV.

In certain embodiments, the antibody of the present inventionspecifically binds to integrin α_(v)β₃. In some embodiments, theantibody comprises the amino acid sequence of the V_(H) and V_(L) chainsof MEDI-522 (Vitaxin®). In other embodiments, the antibody comprises theamino acid sequence of the CDRs of the V_(H) and V_(L) chains ofMEDI-522 (Vitaxin®).

In certain embodiments, the antibody of the present inventionspecifically binds to CD2. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of siplizumab. Inother embodiments, the antibody comprises the amino acid sequence of thecomplementarity determining regions (CDRs) of V_(H) and V_(L) chains ofsiplizumab.

In certain embodiments, the antibody of the present inventionspecifically binds to CD19. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of MT103. In otherembodiments, the antibody comprises the amino acid sequence of the CDRsof the V_(H) and V_(L) chains of MT103.

In certain embodiments, the antibody of the present inventionspecifically binds to an Eph receptor. In certain embodiments, theantibody of the present invention specifically binds to EphA2. In someembodiments, the antibody comprises the amino acid sequence of the CDRsof the V_(H) and V_(L) chains of EA2 or EA5. In other embodiments, theantibody comprises the amino acid sequence of the V_(H) and V_(L) chainsof EA2 or EA5. In certain embodiments, the antibody of the presentinvention specifically binds to EphA4. In some embodiments, the antibodyof the present invention specifically binds to EphB4.

In certain embodiments, the antibody of the present inventionspecifically binds to IL-9. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of MEDI-528. Inother embodiments, the antibody comprises the amino acid sequence of theCDRs of the V_(H) and V_(L) chains of MEDI-528.

Particular antibodies comprising at least one thioether cross-link aredescribed in detail in Section 5.4 below.

In a further aspect, the present invention provides a compositionsubstantially free of a denaturing reagent comprising a population ofantibodies, i.e., two, three, four, five or more antibodies, wherein atleast 2%, 5%, 10%, 15% or 20% of the antibodies comprise at least onethioether cross-link. In some embodiments, about 2-20%, 2-15%, 2-10%,2-5%, 5-20%, 10-15%, 10-30%, 20-30%, 10-40%, 10-50%, 20-50% or 10-75% ofthe antibodies comprise at least one thioether cross-link.

In certain embodiments, at least 2%, 5%, 10%, 15% or 20% of theantibodies comprise at least two thioether cross-link. In someembodiments, about 2-20%, 2-15%, 2-10%, 2-5%, 5-20%, 10-15%, 10-30%,20-30%, 10-40%, 10-50%, 20-50% or 10-75% of the antibodies comprise atleast two thioether cross-link.

In certain embodiments, the composition is a pharmaceutical composition.In some embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carriers.

In certain embodiments, populations of antibodies all have the sameamino acid sequence of V_(H) and V_(L) chains. In some embodiments,populations of antibodies all have the same CDRs of V_(H) and V_(L)chains.

In certain embodiments, populations of antibodies have different aminoacid sequence of V_(H) and V_(L) chains. In some embodiments,populations of antibodies have different CDRs of V_(H) and V_(L) chains.

The present invention provides a composition comprising a fusionprotein. In some embodiments, the fusion protein comprises an Fc domainof an antibody or a fragment thereof, wherein the Fc domain or Fc domainfragment comprises at least one thioether cross-link. In otherembodiments, the fusion protein comprises an C_(H)1, C_(H)2, C_(H)3and/or C_(L) domain of an antibody, wherein the C_(H)1, C_(H)2, C_(H)3or C_(L) domain comprises at least one thioether cross-link. In anotherembodiments, the fusion protein comprises two, three or all of thedomains of C_(H)1, C_(H)2, C_(H)3 or C_(L).

The present invention provides a composition substantially free of adenaturing reagent, the composition comprising a population of fusionproteins, i.e., two, three, four, five or more fusion proteins, whereinat least 2%, 5%, 10%, 15% or 20% of the fusion proteins comprise atleast one thioether cross-link. In certain embodiments, the fusionproteins comprises an Fc domain or a fragment thereof, wherein thethioether cross-link is in the Fc domain or Fc domain fragment. In otherembodiments, the fusion proteins comprise a C_(H)1, C_(H)2, C_(H)3and/or C_(L) domain, wherein the domain comprises the thioethercross-link.

The present invention provides a composition substantially free of adenaturing reagent, the composition comprising a population ofantibodies, i.e., two, three, four, five or more antibodies, whereinless than 2%, 1.5%, 1% or 0.5% of the antibodies in the populationcomprise a thioether cross-link. In certain embodiments, the populationof antibodies have the same amino acid sequences. In other embodiments,the population of antibodies have different amino acid sequences. In aspecific embodiment, the composition is a pharmaceutical composition.

The present invention provides a composition substantially free of adenaturing reagent, the composition comprising a population of fusionproteins, i.e., two, three, four, five or more fusion proteins, whereinless than 2%, 1.5%, 1% or 0.5% of the fusion proteins in the populationcomprise a thioether cross-link. In some embodiments, the fusionproteins comprise an Fc domain or a fragment thereof, wherein thethioether cross-link is in the Fc domain or Fc domain fragment. In otherembodiments, the fusion proteins comprise a C_(H)1, C_(H)2, C_(H)3and/or C_(L) domain, wherein the domain comprises the thioethercross-link. In certain embodiments, the population of antibodies havethe same amino acid sequences. In other embodiments, the population offusion proteins have different amino acid sequences. In a specificembodiment, the composition is a pharmaceutical composition.

5.3.1 Pharmaceutical Compositions and Formulations

The invention provides compositions comprising macromolecules, forinstance, polypeptides (e.g., an antibody or a fusion protein) of theinvention for use in diagnosing, detecting, or monitoring a disorder, inpreventing, treating, managing, or ameliorating of a disorder or one ormore symptoms thereof, and/or in research. In a specific embodiment, acomposition comprises one or more antibodies or fusion proteins of theinvention. In another embodiment, a composition comprises one or moreantibodies fusion proteins of the invention and one or more prophylacticor therapeutic agents other than antibodies fusion proteins of theinvention. Preferably, the prophylactic or therapeutic agents known tobe useful for or having been or currently being used in the prevention,treatment, management, or amelioration of a disorder or one or moresymptoms thereof. In accordance with these embodiments, the compositionmay further comprise of a carrier, diluent or excipient.

The compositions of the invention include, but are not limited to, bulkdrug compositions useful in the manufacture of pharmaceuticalcompositions (e.g., impure or non-sterile compositions) andpharmaceutical compositions (i.e., compositions that are suitable foradministration to a subject or patient) which can be used in thepreparation of unit dosage forms. Such compositions comprise aprophylactically or therapeutically effective amount of a prophylacticand/or therapeutic agent disclosed herein or a combination of thoseagents and a pharmaceutically acceptable carrier. Preferably,compositions of the invention are pharmaceutical compositions andcomprise an effective amount of one or more antibodies of the invention,a pharmaceutically acceptable carrier, and, optionally, an effectiveamount of another prophylactic or therapeutic agent.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete)), excipient, or vehicle with which thetherapeutic is contained in or administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like.

The pharmaceutical composition can be formulated as an oral or non-oraldosage form, for immediate or extended release. The composition cancomprise inactive ingredients ordinarily used in pharmaceuticalpreparation such as diluents, fillers, disintegrants, sweeteners,lubricants and flavors. The pharmaceutical composition is preferablyformulated for intravenous administration, either by bolus injection orsustained drip, or for release from an implanted capsule. A typicalformulation for intravenous administration utilizes physiological salineas a diluent.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The composition of the present invention can also include printed matterthat describes clinical indications for which the antibodies can beadministered as a therapeutic agent, dosage amounts and schedules,and/or contraindications for administration of the antibodies of theinvention to a patient.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents, or pharmaceutical compositions ofthe invention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. Preferably, one or more of the prophylactic or therapeuticagents or pharmaceutical compositions of the invention is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,at least 75 mg, or at least 100 mg. The lyophilized prophylactic ortherapeutic agents or pharmaceutical compositions of the inventionshould be stored at between 2° C. and 8° C. in its original containerand the prophylactic or therapeutic agents, or pharmaceuticalcompositions of the invention should be administered within 1 week,preferably within 5 days, within 72 hours, within 48 hours, within 24hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours,or within 1 hour after being reconstituted. In an alternativeembodiment, one or more of the prophylactic or therapeutic agents orpharmaceutical compositions of the invention is supplied in liquid formin a hermetically sealed container indicating the quantity andconcentration of the agent. Preferably, the liquid form of theadministered composition is supplied in a hermetically sealed containerat least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, atleast 75 mg/ml or at least 100 mg/ml. The liquid form should be storedat between 2° C. and 8° C. in its original container.

Generally, the ingredients of the compositions of the invention arederived from a subject that is the same species origin or speciesreactivity as recipient of such compositions. Thus, in a preferredembodiment, human or humanized antibodies are administered to a humanpatient for therapy or prophylaxis.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection.

The compositions of the invention may be administered topically. Suchcompositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity preferablygreater than water are typically employed. Suitable formulationsinclude, without limitation, solutions, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like, which are, ifdesired, sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, preferably in combination with a solid or liquidinert carrier, is packaged in a mixture with a pressurized volatile(e.g., a gaseous propellant, such as freon) or in a squeeze bottle.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well-known in the art.

The compositions of the invention may be administered intranasally. Suchcomposition can be formulated in an aerosol form, spray, mist or in theform of drops. In particular, prophylactic or therapeutic agents for useaccording to the present invention can be conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant (e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compositions of the invention may be administered orally. Suchcompositions can be formulated orally in the form of tablets, capsules,cachets, gelcaps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The compositions of the invention may be used for pulmonaryadministration, e.g., by use of an inhaler or nebulizer. Suchcompositions can be formulated with an aerosolizing agent. See, e.g.,U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064,5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, eachof which is incorporated herein by reference their entireties. In aspecific embodiment, macromolecules of the invention, combinationtherapy, and/or composition of the invention is administered usingAlkermes AIR™ pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass.).

The compositions of the invention may be formulated for parenteraladministration by injection (e.g., by bolus injection or continuousinfusion). Formulations for injection may be presented in unit dosageform (e.g., in ampoules or in multi-dose containers) with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle (e.g., sterile pyrogen-free water)before use.

The compositions of the invention can be formulated as depotpreparations. Such long acting formulations may be administered byimplantation (e.g., subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compositions may beformulated with suitable polymeric or hydrophobic materials (e.g., as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives (e.g., as a sparingly soluble salt).

5.3.2 Methods of Administration

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a prophylactic or therapeutic agent of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidurala administration, intratumoral administration,and mucosal administration (e.g., intranasal and oral routes). Inaddition, pulmonary administration can be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. See,e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272,5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos.WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, fora description of pulmonary administration, each of which is incorporatedherein by reference their entireties. In one embodiment, an antibody ofthe invention, combination therapy, or a composition of the invention isadministered using Alkermes AIR™ pulmonary drug delivery technology(Alkermes, Inc., Cambridge, Mass.). In a specific embodiment,prophylactic or therapeutic agents of the invention are administeredintramuscularly, intravenously, intratumorally, orally, intranasally,pulmonary, or subcutaneously. The prophylactic or therapeutic agents maybe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention is administered locally to the affected area to a subject toprevent, treat, manage, and/or ameliorate a disorder or a symptomthereof. In another embodiment, an effective amount of one or moreantibodies of the invention is administered locally to the affected areain combination with an effective amount of one or more therapies (e.g.,one or more prophylactic or therapeutic agents) other than an antibodyof the invention of a subject to prevent, treat, manage, and/orameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent can bedelivered in a controlled release or sustained release system. In oneembodiment, a pump may be used to achieve controlled or sustainedrelease (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used to achieve controlled or sustained release of the therapies ofthe invention (see e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Press., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377;

U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No.5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; andPCT Publication No. WO 99/20253. Examples of polymers used in sustainedrelease formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a preferredembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable. In yet another embodiment, a controlled or sustainedrelease system can be placed in proximity of the prophylactic ortherapeutic target, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., 1996, “Intratumoral Radioimmunotherapy of a Human ColonCancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. R^(e1). Bioact. Mater. 24:853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control R^(e1). Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

5.3.3 Dosage and Frequency of Administration

The amount of a prophylactic or therapeutic agent or a composition ofthe present invention which will be effective in the treatment,management, prevention, or amelioration of a disorder or one or moresymptoms thereof can be determined by standard clinical. The frequencyand dosage will vary according to factors specific for each patientdepending on the specific therapy or therapies (e.g., the specifictherapeutic or prophylactic agent or agents) administered, the severityof the disorder, disease, or condition, the route of administration, aswell as age, body, weight, response, the patient's immune status, andthe past medical history of the patient. For example, the dosage of aprophylactic or therapeutic agent or a composition of the inventionwhich will be effective in the treatment, prevention, management, oramelioration of a disorder or one or more symptoms thereof can bedetermined by administering the composition to an animal model such as,e.g., the animal models disclosed herein or known to those skilled inthe art. In addition, in vitro assays may optionally be employed to helpidentify optimal dosage ranges. Suitable regimens can be selected by oneskilled in the art by considering such factors and by following, forexample, dosages reported in the literature and recommended in thePhysician's Desk Reference (57th ed., 2003).

The toxicity and/or efficacy of the prophylactic and/or therapeuticprotocols of the present invention can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Therapies that exhibit large therapeutic indices are preferred. Whiletherapies that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such agents to the siteof affected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any therapy used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

For macromolecules comprising at least one thioesther cross-link of thepresent invention, the dosage administered to a patient is typically0.01 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human and humanized antibodies have alonger half-life within the human body than antibodies from otherspecies due to the immune response to the foreign polypeptides. Thus,lower dosages of human antibodies and less frequent administration isoften possible.

Exemplary doses of a small molecule include milligram or microgramamounts of the small molecule per kilogram of subject or sample weight(e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram).

The dosages of prophylactic or therapeutically agents are described inthe Physicians' Desk Reference (56th ed., 2002).

5.4. ANTIBODIES OF THE PRESENT INVENTION

The antibodies of the present invention include, but are not limited to,monoclonal antibodies, synthetic antibodies, multispecific antibodies(including bi-specific antibodies), human antibodies, humanizedantibodies, chimeric antibodies, single-chain Fvs (scFv) (includingbi-specific scFvs), single chain antibodies, Fab fragments, F(ab′)fragments, disulfide-linked Fvs (sdFv), and epitope-binding fragments ofany of the above. In particular, antibodies of the present inventioninclude immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that immunospecifically binds to an antigen. Theimmunoglobulin molecules of the invention can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass of immunoglobulin molecule. Preferably, theantibodies of the invention are IgG, more preferrably, IgG₁.

In certain embodiments, the antibodies of the invention comprise fourpolypeptide chains—two light chains and two heavy chains. In otherembodiments, the antibodies of the invention comprise a V_(H) chainand/or a V_(L) chain. In yet another embodiments, the antibodies of thepresent invention are epitope-binding fragments.

Throughout the present specification, the numbering of the residues inan IgG light or heavy chain is that of the EU index as in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, NH1, MD (1991), expressly incorporated herein by references.The “EU index as in Kabat” refers to the numbering of the human IgG₁ EUantibody.

The antibodies of the invention may be from any animal origin includingbirds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat,guinea pig, camel, horse, or chicken). Preferably, the antibodies arehuman or humanized monoclonal antibodies. As used herein, “human”antibodies include antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from mice or other animal that express antibodies fromhuman genes.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies mayimmunospecifically bind to different epitopes of a polypeptide or mayimmunospecifically bind to both a polypeptide as well a heterologousepitope, such as a heterologous polypeptide or solid support material.See, e.g., International Publication Nos. WO 93/17715, WO 92/08802, WO91/00360, and WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60-69;U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and5,601,819; and Kostelny et al., 1992, J. Immunol. 148:1547-1553.

The antibodies of the invention include derivatives of the antibodiesknown to those of skill in the art. Standard techniques known to thoseof skill in the art can be used to introduce mutations in the nucleotidesequence encoding an antibody to be used with the methods of theinvention, including, for example, site-directed mutagenesis andPCR-mediated mutagenesis which result in amino acid substitutions.Preferably, the derivatives include less than 25 amino acidsubstitutions, less than 20 amino acid substitutions, less than 15 aminoacid substitutions, less than 10 amino acid substitutions, less than 5amino acid substitutions, less than 4 amino acid substitutions, lessthan 3 amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original molecule. In a preferred embodiment, thederivatives have conservative amino acid substitutions are made at oneor more predicted non-essential amino acid residues. A “conservativeamino acid substitution” is one in which the amino acid residue isreplaced with an amino acid residue having a side chain with a similarcharge. Families of amino acid residues having side chains with similarcharges have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Alternatively, mutations can be introduced randomly alongall or part of the coding sequence, such as by saturation mutagenesis,and the resultant mutants can be screened for biological activity toidentify mutants that retain activity. Following mutagenesis, theencoded protein can be expressed and the activity of the protein can bedetermined.

The antibodies of the present invention include derivatives that aremodified, i.e, by the covalent attachment of any type of molecule to theantibody such that covalent attachment. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, synthesis in the presence oftunicamycin, etc. Additionally, the derivative may contain one or morenon-classical amino acids.

The present invention also provides antibodies of the invention thatcomprise a framework region known to those of skill in the art. Incertain embodiments, one or more framework regions, preferably, all ofthe framework regions, of an antibody to be used in the methods of theinvention or fragment thereof are human. In certain other embodiments ofthe invention, the fragment region of an antibody of the invention ishumanized. In certain embodiments, the antibody to be used with themethods of the invention is a synthetic antibody, a monoclonal antibody,an intrabody, a chimeric antibody, a human antibody, a humanizedchimeric antibody, a humanized antibody, a glycosylated antibody, amultispecific antibody, a human antibody, a single-chain antibody, or abispecific antibody.

In certain embodiments of the invention, the antibodies of the inventionhave half-lives in a mammal, preferably a human, of greater than 12hours, greater than 1 day, greater than 3 days, greater than 6 days,greater than 10 days, greater than 15 days, greater than 20 days,greater than 25 days, greater than 30 days, greater than 35 days,greater than 40 days, greater than 45 days, greater than 2 months,greater than 3 months, greater than 4 months, or greater than 5 months.Antibodies or antigen-binding fragments thereof having increased in vivohalf-lives can be generated by techniques known to those of skill in theart. For example, antibodies or antigen-binding fragments thereof withincreased in vivo half-lives can be generated by modifying (e.g.,substituting, deleting or adding) amino acid residues identified asinvolved in the interaction between the Fc domain and the FcRn receptor(see, e.g., PCT Publication No. WO 97/34631, U.S. patent applicationSer. No. 10/020,354, entitled “Molecules with Extended Half-Lives,Compositions and Uses Thereof”, filed Dec. 12, 2001, by Johnson et al.,and U.S. patent application Ser. No. 11/263,230, filed Oct. 31, 2005,entitled “Methods of Preventing and Treating RSV Infections and RelatedConditions,” by Losonsky, which are incorporated herein by reference intheir entireties). Such antibodies or antigen-binding fragments thereofcan be tested for binding activity to an antigens as well as for in vivoefficacy using methods known to those skilled in the art, for example,by immunoassays described herein.

Further, antibodies with increased in vivo half-lives can be generatedby attaching to said antibodies or antibody fragments polymer moleculessuch as high molecular weight polyethyleneglycol (PEG). PEG can beattached to said antibodies with or without a multifunctional linkereither through site-specific conjugation of the PEG to the N- orC-terminus of said antibodies or via epsilon-amino groups present onlysine residues. Linear or branched polymer derivatization that resultsin minimal loss of biological activity will be used. The degree ofconjugation will be closely monitored by SDS-PAGE and mass spectrometryto ensure proper conjugation of PEG molecules to the antibodies.Unreacted PEG can be separated from antibody-PEG conjugates by, e.g.,size exclusion or ion-exchange chromatography. PEG-derivatizatedantibodies or antigen-binding fragments thereof can be tested forbinding activity to RSV antigens as well as for in vivo efficacy usingmethods known to those skilled in the art, for example, by immunoassaysdescribed herein.

The antibodies of the invention can be single-chain antibodies. Thedesign and construction of a single-chain antibody is described inMarasco et al, 1993, Proc Natl Acad Sci 90:7889-7893, which isincorporated herein by reference in its entirety.

In certain embodiments, the antibodies of the invention bind to anintracellular epitope, i.e., are intrabodies. An intrabody comprises atleast a portion of an antibody that is capable of immunospecificallybinding an antigen and preferably does not contain sequences coding forits secretion. Such antibodies will bind its antigen intracellularly. Inone embodiment, the intrabody comprises a single-chain Fv (“sFv”).

In a further embodiment, the intrabody preferably does not encode anoperable secretory sequence and thus remains within the cell (seegenerally Marasco, W A, 1998, “Intrabodies: Basic Research and ClinicalGene Therapy Applications” Springer: New York).

sFv are antibody fragments comprising the V_(H) and V_(L) domains ofantibody,

wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains which enables the sFv to form thedesired structure for antigen binding. For a review of sFv see Pluckthunin The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg andMoore eds. Springer-Verlag, New York, pp. 269-315 (1994).

5.4.1 Antibody Conjugates

The present invention also encompasses antibodies that are conjugated orfused to one or more moieties, including but not limited to, peptides,polypeptides, proteins, fusion proteins, nucleic acid molecules, smallmolecules, mimetic agents, synthetic drugs, inorganic molecules, andorganic molecules.

The present invention encompasses antibodies that are recombinantlyfused or chemically conjugated (including both covalent and non-covalentconjugations) to a heterologous protein or polypeptide (or fragmentthereof, preferably to a polypeptide of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids) to generate fusionproteins. The fusion does not necessarily need to be direct, but mayoccur through linker sequences. For example, antibodies may be used totarget heterologous polypeptides to particular cell types, either invitro or in vivo, by fusing or conjugating the antibodies to antibodiesspecific for particular cell surface receptors. Antibodies fused orconjugated to heterologous polypeptides may also be used in in vitroimmunoassays and purification methods using methods known in the art.See e.g., International publication No. WO 93/21232; European Patent No.EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat.No. 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et al.,1991, J. Immunol. 146:2446-2452, which are incorporated by reference intheir entireties.

The present invention further includes compositions comprisingheterologous proteins, peptides or polypeptides fused or conjugated toantibody fragments. For example, the heterologous polypeptides may befused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)₂fragment, a Fc domain, a VH domain, a VL domain, a VH CDR, a VL CDR, orfragment thereof. Methods for fusing or conjugating polypeptides toantibody portions are well-known in the art. See, e.g., U.S. Pat. Nos.5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946;European Patent Nos. EP 307,434 and EP 367,166; Internationalpublication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991,Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J.Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA89:11337-11341, (said references incorporated by reference in theirentireties). See also PCT Publication No. WO 97/34631, U.S. patentapplication Ser. No. 10/020,354, entitled “Molecules with ExtendedHalf-Lives, Compositions and Uses Thereof”, filed Dec. 12, 2001, byJohnson et al., and U.S. patent application Ser. No. 11/263,230, filedOct. 31, 2005, entitled “Methods of Preventing and Treating RSVInfections and Related Conditions,” by Losonsky, the contents of whichare incorporated by reference in their entireties.

In certain embodiments, the present invention provides a fusion proteinthat comprises an Fc domain of an antibody or a fragment thereof,wherein the Fc domain or the Fc domain fragment comprises at least onethioether cross-link. Such a fusion protein can be any fusion proteincomprising an Fc domain or an Fc domain fragment known in the art, suchas human tumor necrosis factor receptor Fc fusion protein, as describedin Moreland et al., 2000, New Eng. J. Med. 343:15869-93; or B7.1 Fcfusion protein, as described in Liu et al., 2005 , Cancer Research11(23):8492-8502, the contents of which are incorporated by reference intheir entireties.

In some embodiments, the Fc domain may further comprise one or moreamino acid substitutions (Fc variants). In some embodiments, Fc variantsexhibit altered binding affinity for at least one or more Fc ligands(e.g., FcγRs, c1q). Exemplary Fc variants and methods of making such aredescribed for example, in U.S. Patent Publication Nos. 2006/0039904 and2006/0040325, both published on Feb. 23, 2006, the contents of which areincorporated by reference in their entireties.

In certain embodiments, the present invention provides a fusion proteinthat comprises an C_(H)1, C_(H)2, C_(H)3 and/or C_(L) domain of anantibody, wherein the C_(H)1, C_(H)2, C_(H)3 or C_(L) domain comprisesat least one thioether cross-link.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol.287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313(each of these patents and publications are hereby incorporated byreference in its entirety). Antibodies or fragments thereof, or theencoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. One or more portionsof a polynucleotide encoding an antibody or antibody fragment may berecombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies can be fused to marker sequences, such as apeptide to facilitate purification. In embodiments, the marker aminoacid sequence is a hexa-histidine peptide, such as the tag provided in apQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the hemagglutinin “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., 1984,Cell 37:767) and the “flag” tag.

In other embodiments, antibodies of the present invention, analogs orderivatives thereof can be conjugated to a diagnostic or detectableagent. Such antibodies can be useful for monitoring or prognosing thedevelopment or progression of a disorder as part of a clinical testingprocedure, such as determining the efficacy of a particular therapy.Such diagnosis and detection can be accomplished by coupling theantibody to detectable substances including, but not limited to variousenzymes, such as but not limited to horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; prostheticgroups, such as but not limited to streptavidin/biotin andavidin/biotin; fluorescent materials, such as but not limited to,umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as but not limitedto iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium(³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In,), and technetium (⁹⁹Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and¹¹⁷Tin; positron emitting metals using various positron emissiontomographies, noradioactive paramagnetic metal ions, and molecules thatare radiolabelled or conjugated to specific radioisotopes.

The present invention further encompasses antibodies that are conjugatedto a therapeutic moiety. An antibody or fragment thereof may beconjugated to a therapeutic moiety such as a cytotoxin, e.g., acytostatic or cytocidal agent, a therapeutic agent or a radioactivemetal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includesany agent that is detrimental to cells. Therapeutic moieties include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunoribicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), Auristatin molecules (e.g., auristatin PHE,bryostatin 1, and solastatin 10; see Woyke et al., Antimicrob. AgentsChemother. 46:3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother.45:3580-4 (2001), Mohammad et al., Anticancer Drugs 12:735-40 (2001),Wall et al., Biochem. Biophys. Res. Commun. 266:76-80 (1999), Mohammadet al., Int. J. Oncol. 15:367-72 (1999), all of which are incorporatedherein by reference), hormones (e.g., glucocorticoids, progestins,androgens, and estrogens), DNA-repair enzyme inhibitors (e.g., etoposideor topotecan), kinase inhibitors (e.g., compound ST1571, imatinibmesylate (Kantaijian et al., Clin Cancer Res. 8(7):2167-76 (2002)),cytotoxic agents (e.g., paclitaxel, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof) and those compounds disclosedin U.S. Pat. Nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242,6,242,196, 6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877,5,958,769, 5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904,5,840,745, 5,728,868, 5,648,239, 5,587,459), farnesyl transferaseinhibitors (e.g., R115777, BMS-214662, and those disclosed by, forexample, U.S. Pat. Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960,6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581,6,399,615, 6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765,6,342,487, 6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140,6,232,338, 6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193,6,187,786, 6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366,6,124,465, 6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870,6,077,853, 6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582,6,051,574, and 6,040,305), topoisomerase inhibitors (e.g., camptothecin;irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI 147211);DX-895 If; IST-622; rubitecan; pyrazoloacridine; XR-5000; saintopin;UCE6; UCE1022; TAN-1518A; TAN-1518B; KT6006; KT6528; ED-110; NB-506;ED-110; NB-506; and rebeccamycin); bulgarein; DNA minor groove binderssuch as Hoescht dye 33342 and Hoechst dye 33258; nitidine; fagaronine;epiberberine; coralyne; beta-lapachone; BC-4-1; bisphosphonates (e.g.,alendronate, cimadronte, clodronate, tiludronate, etidronate,ibandronate, neridronate, olpandronate, risedronate, piridronate,pamidronate, zolendronate) HMG-CoA reductase inhibitors, (e.g.,lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, statin,cerivastatin, lescol, lupitor, rosuvastatin and atorvastatin) andpharmaceutically acceptable salts, solvates, clathrates, and prodrugsthereof. See, e.g., Rothenberg, M. L., Annals of Oncology 8:837-855(1997); and Moreau, P., et al., J. Med. Chem. 41:1631-1640 (1998)),antisense oligonucleotides (e.g., those disclosed in the U.S. Pat. Nos.6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709),immunomodulators (e.g., antibodies and cytokines), antibodies, andadenosine deaminase inhibitors (e.g., Fludarabine phosphate and2-Chlorodeoxyadenosine).

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety or drug moiety that modifies a given biologicalresponse. Therapeutic moieties or drug moieties are not to be construedas limited to classical chemical therapeutic agents. For example, thedrug moiety may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, cholera toxin, ordiphtheria toxin; a protein such as tumor necrosis factor,.alpha.-interferon, .beta.-interferon, nerve growth factor, plateletderived growth factor, tissue plasminogen activator, an apoptotic agent,e.g., TNFα, TNFβ, AIM I (see, International publication No. WO97/33899), AIM II (see, International Publication No. WO 97/34911), FasLigand (Takahashi et al., 1994, J. Immunol., 6:1567-1574), and VEGI(see, International publication No. WO 99/23105), a thrombotic agent oran anti-angiogenic agent, e.g., angiostatin, endostatin or a componentof the coagulation pathway (e.g., tissue factor); or, a biologicalresponse modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), a growth factor (e.g.,growth hormone (“GH”)), or a coagulation agent (e.g., calcium, vitaminK, tissue factors, such as but not limited to, Hageman factor (factorXII), high-molecular-weight kininogen (HMWK), prekallikrein (PK),coagulation proteins-factors II (prothrombin), factor V, XIIa, VIII,XIIIa, XI, XIa, IX, IXa, X, phospholipid, fibrinopeptides A and B fromthe α and P chains of fibrinogen, fibrin monomer).

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive metal ion, such as alph-emitters such as ²¹³Bi ormacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, ¹³¹In, ¹³¹LU, ¹³¹Y, ¹³¹Ho, ¹³¹Sm, to polypeptides.In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraa-cetic acid (DOTA)which can be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug.Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol.26(8):943-50, each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

The therapeutic moiety or drug conjugated to an antibody or fragmentthereof should be chosen to achieve the desired prophylactic ortherapeutic effect(s) for a particular disorder in a subject. Aclinician or other medical personnel should consider the following whendeciding on which therapeutic moiety or drug to conjugate to an antibodyor fragment thereof: the nature of the disease, the severity of thedisease, and the condition of the subject.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

5.4.2 Isolated Antibodies Comprising at Least One Thioether Cross-Linkthat Specifically Bind to a Particular Antigen of and CompositionsComprising the Same

In further embodiments, the present invention provides isolatedantibodies or compositions comprising antibodies, wherein saidantibodies comprises at least one thioether cross-link, and wherein saidantibodies specifically bind to one or more particular antigens.

In certain embodiments, the antibody of the present inventionspecifically binds to an antigen of respiratory syncytial virus (RSV).In some embodiments, the antibody comprises the amino acid sequence ofthe variable heavy (V_(H)) and variable light (VL) chains of palivizumabor motavizumab. In other embodiments, the antibody comprises the aminoacid sequence of the complementarity determining regions (CDRs) of theV_(H) and V_(L) chains of palivizumab or motavizumab.

In certain embodiments, the antibody of the present inventionspecifically binds to an antigen of human metapneumovirus (hMPV). Insome embodiments, the antibody is a humanized antibody that specificallybinds to an antigen of hMPV.

In certain embodiments, the antibody of the present inventionspecifically binds to integrin α_(v)β₃. In some embodiments, theantibody comprises the amino acid sequence of the V_(H) and V_(L) chainsof MEDI-522 (Vitaxin®). In other embodiments, the antibody comprises theamino acid sequence of the CDRs of the V_(H) and V_(L) chains ofMEDI-522 (Vitaxin®).

In certain embodiments, the antibody of the present inventionspecifically binds to CD2. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of siplizumab. Inother embodiments, the antibody comprises the amino acid sequence of thecomplementarity determining regions (CDRs) of V_(H) and V_(L) chains ofsiplizumab.

In certain embodiments, the antibody of the present inventionspecifically binds to CD19. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of MT103. In otherembodiments, the antibody comprises the amino acid sequence of the CDRsof the V_(H) and V_(L) chains of MT103.

In certain embodiments, the antibody of the present inventionspecifically binds to an Eph receptor. In certain embodiments, theantibody of the present invention specifically binds to EphA2. In someembodiments, the antibody comprises the amino acid sequence of the V_(H)and V_(L) chains of EA2 or EA5. In other embodiments, the antibodycomprises the amino acid sequence of the CDRs of the V_(H) and V_(L)chains of EA2 or EA5. In certain embodiments, the antibody of thepresent invention specifically binds to EphA4. In some embodiments, theantibody of the present invention specifically binds to EphB4.

In certain embodiments, the antibody of the present inventionspecifically binds to IL-9. In some embodiments, the antibody comprisesthe amino acid sequence of the V_(H) and V_(L) chains of MEDI-528. Inother embodiments, the antibody comprises the amino acid sequence of theCDRs of the V_(H) and V_(L) chains of MEDI-528.

In certain embodiments, the antibodies of the present invention arethose antibodies other than an antibody that specifically binds to anantigen of RSV, an antigen of human metapneumovirus (hMPV), integrinα_(v)β₃, CD2, CD19, Eph receptor (e.g., EphA2, EphA4 or EphB4), or IL-9.In certain embodiments, the antibody of the present invention is not anantibody that specifically binds to an antigen of respiratory syncytialvirus (RSV). In other embodiments, the antibody of the present inventionis not an antibody that specifically binds to an antigen of humanmetapneumovirus (hMPV). In certain embodiments, the antibody of thepresent invention is not an antibody that specifically binds to integrinα_(v)β₃. In certain embodiments, the antibody of the present inventionis not an antibody that specifically binds to CD2. In certainembodiments, the antibody of the present invention is not an antibodythat specifically binds to CD19. In further embodiments, the antibody ofthe present invention is not an antibody that specifically binds toEphA2. In certain embodiments, the antibody of the present invention isnot an antibody that specifically binds to EphA4. In certainembodiments, the antibody of the present invention is not an antibodythat specifically binds to IL-9.

In some embodiments, the antibody is not palivizumab. In otherembodiments, the antibody is not motavizumab. In some embodiments, theantibody is not MEDI-522 (Vitaxin®). In some embodiments, the antibodyis not siplizumab. In some embodiments, the antibody is not MT-103™. Insome embodiments, the antibody is not human or humanized EA2 or EA5. Insome embodiments, the antibody is not MEDI-528.

The antibodies of the present invention may be high potency antibodies.The term “high potency” as used herein refers to antibodies that exhibithigh potency as determined in various assays for biological activity(e.g., neutralization of an antigen). High potency antibodies can beproduced by genetically engineering appropriate antibody gene sequencesand expressing the antibody sequences in a suitable host. See U.S.Application Publication No. 2002/0098189, published Jul. 25, 2002, thecontents of which are incorporate by reference in their entirety. Theantibodies produced can be screened to identify antibodies with, e.g.,high k_(on) values in a BIAcore assay.

In a specific embodiment, the antibodies of the present invention havean association rate constant or k_(on) rate (antibody (Ab)+antigen(Ag)^(k) ^(on) →Ab−Ag) of at least 10⁵ M⁻¹ s⁻¹, at least 5×10⁵ M⁻¹ s⁻¹,at least 10⁶ M⁻¹ s⁻¹, at least 5×10⁶ M⁻¹ s⁻¹, at least 10⁷ M⁻¹ s⁻¹, atleast 5×10⁷ M⁻¹ s⁻¹, or at least 10⁸ M⁻¹ s⁻¹. In a preferred embodiment,the antibodies of the present invention have a k_(on) of at least 2×10⁵M⁻¹ s⁻¹, at least 5×10⁵ M⁻¹ s⁻¹, at least 10⁶ M⁻¹ s⁻¹, at least 5×10⁶M⁻¹ s⁻¹, at least 10⁷ M⁻¹ s⁻¹, at least 5×10⁷ M⁻¹ s⁻¹, or at least 10⁸M⁻¹ s⁻¹. In some embodiments, the antibodies of the present inventionhave a k_(on) rate between 10⁵ M⁻¹ s⁻¹ and 10⁸ M⁻¹ s⁻¹, between 10⁵ M⁻¹s⁻¹ and 10⁸ M⁻¹ s⁻¹, between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, between 10⁵M⁻¹ s⁻¹ and 10⁶ M⁻¹ s⁻¹, or between 10⁶ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹.

In another embodiment, the antibodies of the present invention have ak_(off) rate (antibody (Ab)+antigen) of less than 5×10⁻¹ s⁻¹, less than10⁻¹ s⁻¹, less than 5×10⁻² s⁻¹, less than 10⁻² s⁻¹, less than 5×10⁻³s⁻¹, less than 10⁻³ s⁻¹, less than 5×10⁻⁴ s⁻¹, less than 10⁻⁴ s⁻¹, lessthan 5×10⁻⁵ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than10⁻⁶ s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁸s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁹ s⁻¹, less than 10⁻⁹ s⁻¹, orless than 10⁻¹⁰ s⁻¹. In a preferred embodiment, the antibodies of thepresent invention have a k_(off) of less than 5×10⁻⁴ s⁻¹, less than5×10⁻⁵ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁶s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁸ s⁻¹,less than 10⁻⁸ s⁻¹, less than 5×10⁻⁹ s⁻¹, less than 10⁻⁹ s⁻¹, or lessthan 10⁻¹⁰ s⁻¹. In some embodiments, the antibodies of the presentinvention have a k_(off) between 5×10⁻⁴ s⁻¹ and 10⁻¹⁰ s⁻¹, between5×10⁻⁴ s⁻¹ and 10⁻⁹ s⁻¹, between 5×10⁻⁴ s⁻¹ and 10⁻⁸ s⁻¹, between 5×10⁻⁷s⁻¹ and 10⁻⁷ s⁻¹, between 10⁻⁵ s⁻¹ and 10⁻¹⁰ s⁻¹, or between 10⁻⁶ s⁻¹and 10⁻⁹ s⁻¹.

In certain embodiments, the antibodies of the present invention have ahigh binding affinity for one or more antigens. See U.S. Pat. No.6,656,467, the contents of which are incorporated by reference in itsentirety. In certain embodiments, the antibodies of the presentinvention have an affinity constant or K_(a) (k_(on)/k_(off)) of atleast 10² M⁻¹, at least 5×10² M⁻¹, at least 10³ M⁻¹, at least 5×10³ M⁻¹,at least 10⁴ M⁻¹, at least 5×10⁴ M⁻¹, at least 10⁵ M⁻¹, at least 5×10⁵M⁻¹, at least 10⁶ M⁻¹, at least 5×10⁶ M⁻¹, at least 10⁷ M⁻¹, at least5×10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 5×10⁸ M⁻¹, at least 10⁹ M⁻¹, atleast 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 5×10¹⁰ M⁻¹, at least 10¹¹M⁻¹, at least 5×10¹¹ M⁻¹, at least 10¹² M⁻¹, at least 5×10¹² M⁻¹, atleast 10¹³ M⁻¹, at least 5×10¹³ M⁻¹, at least 10¹⁴ M⁻¹, at least 5×10¹⁴M⁻¹, at least 10¹⁵ M⁻¹, or at least 5×10¹⁵ M⁻¹. The present inventionalso provides compositions comprising one or more antibodies whichimmunospecifically bind to an antigen with an affinity constant of atleast 2×10⁸ M⁻¹, at least 2.5×10⁸ M⁻¹, at least 5×10⁸ M⁻¹, at least 10⁹M⁻¹, at least 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 5×10¹⁰ M⁻¹, atleast 10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, at least 10¹² M⁻¹, at least 5×10¹²M⁻¹, at least 10¹³ M⁻¹, at least 5×10¹³ M⁻¹, at least 10¹⁴ M⁻¹, at least5×10¹⁴ M⁻¹, at least 10¹⁵ M⁻¹, or at least 5×10¹⁵ M⁻¹. In certainembodiments, the antibodies of the present invention has K_(a)(k_(on)/k_(off)) between 10² M⁻¹ and 5×10¹⁵ M⁻¹, between 10⁴ M⁻¹ and5×10¹⁵ M⁻¹, between 10⁶ M⁻¹ and 5×10¹⁵ M⁻¹, between 10⁸ M⁻¹ and 5×10¹⁵M⁻¹, between 10¹⁰ M⁻¹ and 5×10¹⁵ M⁻¹, between 10¹² M⁻¹ and 5×10¹⁵ M⁻¹,between 10¹⁴ M⁻¹ and 5×10¹⁵ M⁻¹, between 10⁴ M⁻¹ and 10¹⁴ M⁻¹, between10⁶ M⁻¹ and 10¹² M⁻¹, or between 10⁸ M⁻¹ and 10¹⁰ M⁻¹.

In yet another embodiment, the antibodies of the present invention havea dissociation constant or K_(d) (k_(off)/k_(on)) of less than 5×10⁻² M,less than 10⁻² M, less than 5×10⁻³ M, less than 10⁻³ M, 5× less than10⁻⁴ M, less than 10⁻⁴ M, 5× less than 10⁻⁵ M, less than 10⁻⁵ M, lessthan 5×10⁻⁶ M, less than 10⁻⁶ M, less than 5×10⁻⁷ M, less than 10⁻⁷ M,less than 5×10⁻⁸ M, less than 10⁻⁸ M, less than 5×10⁻⁹ M, less than 10⁻⁹M, less than 5×10⁻¹⁰ M, less than 10⁻¹⁰ M, less than 5×10⁻¹¹ M, lessthan 10⁻¹¹ M, less than 5×10⁻¹² M, less than 10⁻¹² M, less than 5×10⁻¹³M, less than 10⁻¹³ M, less than 5×10⁻¹⁴ M, less than 10⁻¹⁴ M, less than5×10⁻¹⁵ M, or less than 10⁻¹⁵ M. In some embodiments, the antibodies ofthe present invention have a K_(d) between 10⁻² M and 5×10⁻¹⁵ M, between10⁻⁵ M and 5×10⁻¹⁵ M, between 10⁻⁸ M and 5×10⁻¹⁵ M, between 10⁻¹¹ M and5×10⁻¹⁵ M, between 10⁻⁴ M and 10⁻¹⁴ M, between 10⁻⁶ M and 10⁻¹² M, orbetween 10⁻⁸ M and 10⁻¹⁰ M.

In certain embodiments, the antibodies of the present invention have amedian effective concentration (EC₅₀) of less than 0.01 nM, less than0.025 nM, less than 0.05 nM, less than 0.1 nM, less than 0.25 nM, lessthan 0.5 nM, less than 0.75 nM, less than 1 nM, less than 1.25 nM, lessthan 1.5 nM, less than 1.75 nM, or less than 2 nM, in an in vitromicroneutralization assay. The median effective concentration is theconcentration of antibody or antibody fragments that neutralizes 50% ofan antigen in an in vitro microneutralization assay. In a preferredembodiment, the antibodies of the present invention have an EC₅₀ of lessthan 0.01 nM, less than 0.025 nM, less than 0.05 nM, less than 0.1 nM,less than 0.25 nM, less than 0.5 nM, less than 0.75 nM, less than 1 nM,less than 1.25 nM, less than 1.5 nM, less than 1.75 nM, or less than 2nM, in an in vitro microneutralization assay. In certain embodiments,the antibodies of the present invention have an EC₅₀ between 0.01 nM and2 nM, between 0.025 nM and 1.75 nM, between 0.05 nM and 1.5 nM, between0.1 nM and 1.25 nM, or between 0.25 nM and 1 nM.

5.4.2.1. Antibodies Comprising at Least One Thioether Cross-Link thatSpecifically Bind to an Antigen of Respiratory Syncytial Virus (RSV) andCompositions Comprising the Same

The present invention provides an isolated antibody that comprises atleast one thioether cross-link and specifically binds to an antigen ofrespiratory syncytial virus (RSV) and a composition comprising thisantibody. The thioether cross-link is described in detail in Section5.2. The antibodies of the present invention can comprise one ormultiple thioether cross-links. The thioether cross-link can link anytwo residues of the antibody. In certain embodiments, the residueslinked by the thioether cross-link are natural residues. In preferredembodiments, two of the residues are cysteine residues. The thioethercross-link can be at any location of the antibodies where feasibleaccording to the knowledge of those of skill in the art. In preferredembodiments, the thioether cross-link links a heavy chain and a lightchain of the antibody. In particular preferred embodiments, thethioether cross-link links a cysteine of a heavy chain and a cysteine ofa light chain of the antibody.

This antibody of the invention specifically binds to an antigen ofrespiratory syncytial virus (RSV). The term “anti-RSV-antigen antibody”refers to an antibody that binds immunospecifically to a RSV antigen. ARSV antigen refers to a RSV polypeptide or fragment thereof such as ofRSV nucleoprotein, RSV phosphoprotein, RSV matrix protein, RSV smallhydrophobic protein, RSV RNA-dependent RSV polymerase, RSV F protein,and RSV G protein. A RSV antigen also refers to a polypeptide that has asimilar amino acid sequence compared to a RSV polypeptide or fragmentthereof such as of RSV nucleoprotein, RSV phosphoprotein, RSV matrixprotein, RSV small hydrophobic protein, RSV RNA-dependent RSVpolymerase, RSV F protein, and RSV G protein. In certain embodiments,the antibody specifically binds to an epitope in the A antigenic site ofthe fusion (F) protein of RSV.

The anti-RSV-antigen antibody of the invention can be a monoclonalantibody, human antibody, humanized antibody or chimeric antibody. Insome preferred embodiments, the present invention provides a palivizumabthat comprises at least one thioether cross-link. In a specificpreferred embodiment, the palivizumab is SYNAGIS®.

Palivizumab is a humanized monoclonal antibody produced by recombinantDNA technology that specifically binds to an epitope in the A antigenicsite of the fusion (F) protein of RSV. It is a composite of human (95%)and murine (5%) antibody sequences. Palivizumab has high specificactivity against RSV in vitro and is known to neutralize a broad rangeof RSV isolates. Since it is not derived from human plasma, prophylactictreatment with palivizumab does not carry potential risk of transmissionof blood borne pathogens. The amino acid sequence of palivizumab isdisclosed, e.g., in Johnson et al., 1997, J. Infectious Disease176:1215-1224, and International Application Publication No.: WO02/43660, entitled “Methods of Administering/Dosing Anti-RSV Antibodiesfor Prophylaxis and Treatment”, by Young et al., which are incorporatedherein by reference in their entireties. The properties and uses ofpalivizumab are also disclosed in, e.g., other applications, see, e.g.,U.S. patent application Ser. No. 09/724,396 filed Nov. 28, 2000; U.S.patent application Ser. No. 09/996,265 filed Nov. 28, 2001 and U.S.patent application Ser. No. 10/403,180 filed Mar. 31, 2003, all of whichare incorporated herein by reference.

In some embodiments, the antibody comprises the amino acid sequence ofthe V_(H) and V_(L) chains of palivizumab. In other embodiments, theantibody comprises the amino acid sequence of the CDRs of the V_(H) andV_(L) chains of palivizumab. The amino acid sequences of the V_(H) andV_(L) chains of palivizumab and the CDRs of the V_(H) and V_(L) chainsof palivizumab are listed in Table 1 and provided in U.S. patentapplication Ser. No. 11/263,230, filed Oct. 31, 2005, entitled “Methodsof Preventing and Treating RSV Infections and Related Conditions,” byLosonsky, the contents of which are hereby incorporated by reference intheir entirety.

In further embodiments, the invention provides a motavizumab thatcomprises at least one thioether cross-link. The amino acid sequence,properties and uses of motavizumab are disclosed in U.S. Pat. No.6,818,216 and Young et al. In some embodiments, the amino acid sequenceof motavizumab is encoded by SEQ ID NOs. 254 and 255 for the heavy andlight chain respectively, disclosed in U.S. Pat. No. 6,818,216.

In certain specific embodiments, the anti-RSV-antigen antibody is AFFF;P12f2 P12f4; P11d4; A1e9; A12a6; A13c4; A17d4; A4B4; 1X-493L1; FRH3-3F4; M3H9; Y10H6; DG; AFFF(1); 6H8; L1-7E5; L2-15B10; A13a11; A1h5;A4B4(1); A4B4-F52S; or A4B4L1FR-S28R. These antibodies are disclosed inInternational Application Publication No.: WO 02/43660, entitled“Methods of Administering/Dosing Anti-RSV Antibodies for Prophylaxis andTreatment”, by Young et al., and U.S. patent application Ser. No.11/263,230, filed Oct. 31, 2005, entitled “Methods of Preventing andTreating RSV Infections and Related Conditions,” by Losonsky, which isincorporated herein by reference in its entirety.

In some embodiments, the antibody comprises the amino acid sequence ofthe V_(H) and V_(L) chains of motobizumab. In other embodiments, theantibody comprises the amino acid sequence of the CDRs of the V_(H) andV_(L) chains of motobizumab. The amino acid sequences of the V_(H) andV_(L) chains of palivizumab and the CDRs of the V_(H) and V_(L) chainsof motobizumab are listed in Table 1 and provided in U.S. patentapplication Ser. No. 11/263,230, filed Oct. 31, 2005, entitled “Methodsof Preventing and Treating RSV Infections and Related Conditions,” byLosonsky, the contents of which are hereby incorporated by reference intheir entirety.

The anti-RSV-antigen antibodies of this section can be made, formulated,administered, used therapeutically or used prophylactically as describedin U.S. Pat. No. 5,824,307; U.S. Pat. No. 6,818,216; U.S. patentapplication Ser. No. 09/724,396 filed Nov. 28, 2000; U.S. patentapplication Ser. No. 09/724,531 filed Nov. 28, 2000; U.S. patentapplication Ser. No. 09/996,265 filed Nov. 28, 2001; U.S. patentapplication Ser. No. 10/403,180 filed Mar. 31, 2003; U.S. patentapplication Ser. No. 09/796,848 filed Mar. 1, 2001 and published on Jul.25, 2002, as U.S. Pat. Pub. No. 2002/0098189; U.S. patent applicationSer. No. 10/135,636, filed Apr. 29, 2002 and published May 29, 2003, asU.S. Pat. Pub. No. 2002/0097974; U.S. patent application Ser. No.10/461,904 filed Jun. 13, 2003; U.S. patent application Ser. No.10/461,863 filed Jun. 13, 2003 and published on Jan. 29, 2004, as U.S.Pat. Pub. No. 2004/0018200, and U.S. patent application Ser. No.11/263,230, filed October 31, 2005, entitled “Methods of Preventing andTreating RSV Infections and Related Conditions,” by Losonsky, thecontents of which are hereby incorporated by reference in theirentirety.

TABLE 1 VH, VL AND CDR SEQUENCES Antibody VH VH Name Chain Domain VHCDR1 VH CDR2 VH CDR3 palivizumab SEQ ID SEQ ID TSGMSVG DIWWDDKKDYNSMITNWYFDV NO: 1 NO: 2 (SEQ ID NO: 3) PSLKS (SEQ ID NO: 5) (SEQ ID NO:4) motovizumab SEQ ID SEQ ID TAGMSVG DIWWDDKKHYN DMIFNFYFDV NO: 11 NO:12 (SEQ ID NO: 13) PSLKD (SEQ ID NO: 15) (SEQ ID NO: 14) EA2 SEQ ID SEQID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 NO: 19 siplizumab SEQ ID NO: 27SEQ ID NO: 28 SEQ ID NO: 29 7F3com-2H2 SEQ ID NO: 33 SEQ ID NO: 34 SEQID NO: 35 MT-103 ™ CD19 portion (SEQ ID NO: 40) CD3 portion (SEQ ID NO:41) Antibody VL VL Name Chain Domain VL CDR1 VL CDR2 VL CDR3 palivizumabSEQ ID SEQ ID KCQLSVGYMH DTSKLAS FQGSGYPFT NO: 6 NO: 7 (SEQ ID NO: 8)(SEQ ID (SEQ ID NO: 9) NO: 10) motovizumab SEQ ID SEQ ID SASSRVGYMHDTSKLAS FQGSGYPFT NO: 16 NO: 17 (SEQ ID NO: 18) (SEQ ID (SEQ ID NO: 9)NO: 10) EA2 SEQ ID SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 23 NO: 26siplizumab SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 7F3com-2H2 SEQ IDNO: 36 SEQ ID NO: 37 SEQ ID NO: 39 MT-103 ™ CD19 portion SEQ ID NO: 39CD3 portion (SEQ ID NO: 42)

5.4.2.2. Antibodies Comprising at Least One Thioether Cross-Link thatSpecifically Bind to an Antigen of Human Metapneumovirus (hMPV) andCompositions Comprising the Same

The present invention provides an isolated antibody that comprises atleast one thioether cross-link and specifically binds to an antigen ofhuman metapneumovirus (hMPV) and a composition comprising this antibody.The thioether cross-link is described in detail in Section 5.2. Theantibodies of the present invention can comprise one or multiplethioether cross-links. The thioether cross-link can link any tworesidues of the antibody. In certain embodiments, the residues linked bythe thioether cross-link are natural residues. In preferred embodiments,two of the residues are cysteine residues. The thioether cross-link canbe at any location of the antibodies where feasible according to theknowledge of those of skill in the art. In preferred embodiments, thethioether cross-link links a heavy chain and a light chain of theantibody. In particular preferred embodiments, the thioether cross-linklinks a cysteine of a heavy chain and a cysteine of a light chain of theantibody.

This antibody of the invention specifically binds to an antigen of humanmetapneumovirus (hMPV). The term “anti-hMPV-antigen antibody” refers toan antibody or antibody fragment thereof that binds immunospecificallyto a hMPV antigen. A hMPV antigen refers to a hMPV polypeptide orfragment thereof such as of hMPV nucleoprotein, hMPV phosphoprotein,hMPV matrix protein, hMPV small hydrophobic protein, hMPV RNA-dependenthMPV polymerase, hMPV F protein, and hMPV G protein. A hMPV antigen alsorefers to a polypeptide that has a similar amino acid sequence comparedto a hMPV polypeptide or fragment thereof such as of hMPV nucleoprotein,hMPV phosphoprotein, hMPV matrix protein, hMPV small hydrophobicprotein, hMPV RNA-dependent hMPV polymerase, hMPV F protein, and hMPV Gprotein.

The anti-hMPV-antigen antibodies of this invention can be monoclonalantibodies, human antibodies, humanized antibodies or chimericantibodies. In some preferred embodiments, the anti-hMPV antibody of theinvention is the antibody disclosed in U.S. patent application Ser. No.10/628,088, filed Jul. 25, 2003 and published May 20, 2004, as U.S. Pat.Pub. No. US 2004/0096451 A1.

The anti-hMPV-antigen antibodies of this section can be made,formulated, administered, used therapeutically or used prophylacticallyas described in U.S. patent application Ser. No. 10/628,088, filed Jul.25, 2003 and published May 20, 2004, as U.S. Pat. Pub. No. US2004/0096451 A1, the contents of which are hereby incorporated byreference in their entirety.

5.4.2.3. Antibodies Comprising at Least One Thioether Cross-Link thatSpecifically Bind to Integrin α_(v)β₃ and Compositions Comprising theSame

The present invention provides an isolated antibody that comprises atleast one thioether cross-link and specifically binds to integrinα_(v)β₃ and a composition comprising this antibody. The thioethercross-link is described in detail in Section 5.2. The antibodies of thepresent invention can comprise one or multiple thioether cross-links.The thioether cross-link can link any two residues of the antibody. Incertain embodiments, the residues linked by the thioether cross-link arenatural residues. In preferred embodiments, two of the residues arecysteine residues. The thioether cross-link can be at any location ofthe antibodies where feasible according to the knowledge of those ofskill in the art. In preferred embodiments, the thioether cross-linklinks a heavy chain and a light chain of the antibody. In particularpreferred embodiments, the thioether cross-link links a cysteine of aheavy chain and a cysteine of a light chain of the antibody.

This antibody of the invention specifically binds to integrin α_(v)β₃.The antibodies can be monoclonal antibodies, human antibodies, humanizedantibodies or chimeric antibodies. In some preferred embodiments, theanti-integrin α_(v)β₃ antibody of the invention is MEDI-522 (Vitaxin®).Vitaxin® and compositions or formulations comprising Vitaxin® aredisclosed, e.g., in International Publication Nos. WO 98/33919, WO00178815, and WO 02/070007; U.S. application Ser. No. 09/339,222; U.S.patent application Ser. No. 10/091,236, filed Mar. 4, 2002 and publishedNov. 12, 2002, as U.S. Pat. Pub. No. US 2002/0168360, each of which isincorporated herein by reference in its entirety.

In further embodiments, the antibody that immunospecifically binds tointegrin α_(v)β₃ is not Vitaxin® or an antigen-binding fragment ofVitaxin®. Examples of known antibodies that immunospecifically bind tointegrin α_(v)β₃ include, but are not limited to, 11D2 (Searle), themurine monoclonal LM609 (Scripps, International Publication Nos. WO89/05155 and U.S. Pat. No. 5,753,230, which is incorporated herein byreference in its entirety), International Publication Nos WO 98/33919and WO 00/78815, each of which is incorporated herein by reference inits entirety), 17661-37E and 17661-37E1-5 (USBiological), MON 2032 and2033 (CalTag), ab7166 (BV3) and ab 7167 (BV4) (Abcam), and WOW-1(Kiosses et al., Nature Cell Biology, 3:316-320).

α_(v)β₃ an integrin has been found on new blood vessels as well assurface of many solid tumors, activated macrophages, monocytes, andosteoclasts. As the such, the anti- integrin α_(v)β₃ antibodies of thissection can be used, for example, as an investigational antibody, or inthe prevention or treatment of several destructive diseases.

The anti-integrin α_(v)β₃ antibodies of this section can be made,formulated, administered, used therapeutically or used prophylacticallyas described in U.S. patent application Ser. No. 10/091,236, filed Mar.4, 2002 and published Nov. 12, 2002, as U.S. Pat. Pub. No. US2002/0168360; U.S. patent application Ser. No. 10/769,712, filed Jan.30, 2004; U.S. patent application Ser. No. 10/769,720, filed Jan. 30,2004 and published Sep. 9, 2004, as U.S. Pat. Pub. No. US 2004/0176272;U.S. patent application Ser. No. 10/379,145, filed Mar. 4, 2003; U.S.patent application Ser. No. 10/379,189, filed Mar. 4, 2003 and publishedas U.S. Pat. Pub. No. US 2004/0001835; PCT Application No.PCT/US04/02701, filed Jan. 30, 2004; International ApplicationPublication No.: WO 00/78815 A1, entitled “Anti-α_(v)β₃ recombinanthuman antibodies, nucleic acids encoding same and methods”, by Huse etal.; and International Application Publication No.: WO 98/33919 A1,entitled “Anti-alpha-V-veta-3 recombinant humanized antibodies, nucleicacids encoding same and methods of use”, by Huse et al.; InternationalPublication No. WO 89/05155, the contents of which are herebyincorporated by reference in their entirety.

5.4.2.4. Antibodies Comprising at Least One Thioether Cross-Link thatSpecifically Bind to CD2 and Compositions Comprising the Same

The present invention provides an isolated antibody that comprises atleast one thioether cross-link and specifically binds to CD2 and acomposition comprising this antibody. The thioether cross-link isdescribed in detail in Section 5.2. The antibodies of the presentinvention can comprise one or multiple thioether cross-links. Thethioether cross-link can link any two residues of the antibody. Incertain embodiments, the residues linked by the thioether cross-link arenatural residues. In preferred embodiments, two of the residues arecysteine residues. The thioether cross-link can be at any location ofthe antibodies where feasible according to the knowledge of those ofskill in the art. In preferred embodiments, the thioether cross-linklinks a heavy chain and a light chain of the antibody. In particularpreferred embodiments, the thioether cross-link links a cysteine of aheavy chain and a cysteine of a light chain of the antibody.

This antibody of the invention specifically binds to CD2. The antibodiescan be monoclonal antibodies, human antibodies, humanized antibodies orchimeric antibodies. In some preferred embodiments, the anti-CD2antibody of the invention is siplizumab (MEDI-507). Siplizumab canselectively binds to cells expressing the CD2 antigen (specifically Tcells, natural killer cells and thymocytes) and can be used, forexample, in the prophylaxis and treatment of T cell lymphoma or otherrelated conditions. MEDI-507 is disclosed, e.g., in InternationalPublication No. WO 99/03502, International Application Nos.PCT/US02/22273 and PCT/US02/06761, and U.S. application Ser. Nos.09/462,140, 10/091,268, and 10/091,313, each of which is incorporatedherein by reference in its entirety. MEDI-507 is a humanized IgG1κ classmonoclonal antibody that immunospecifically binds to human CD2polypeptide. MEDI-507 was constructed using molecular techniques toinsert the CDRs from the rat monoclonal antibody LO-CD2a/BTI-322 into ahuman IgG1 framework. LO-CD2a/BTI-322 has the amino acid sequencedisclosed, e.g., in U.S. Pat. Nos. 5,730,979, 5,817,311, and 5,951,983;and U.S. application Ser. Nos. 09/056,072 and 09/462,140 (each of whichis incorporated herein by reference in its entirety), or the amino acidsequence of the monoclonal antibody produced by the cell line depositedwith the American Type Culture Collection (ATCC®), 10801 UniversityBoulevard, Manassas, Va. 20110-2209 on Jul. 28, 1993 as Accession NumberHB 11423.

In some embodiments, the antibody comprises the amino acid sequence ofthe V_(H) and V_(L) chains of siplizumab (MEDI-507). In otherembodiments, the antibody comprises the amino acid sequence of the CDRsof the V_(H) and V_(L) chains of siplizumab (MEDI-507). The amino acidsequences of the CDRs of the V_(H) and V_(L) chains of siplizumab(MEDI-507) are listed in Table 1 are provided in U.S. Patent PublicationNo. 2003/0044406, published on Mar. 6, 2003, the contents of which arehereby incorporated by reference in its entirety.

The anti-CD2 antibodies of this section can be made, formulated,administered, used therapeutically or prophylactically, or in othercontext as described in U.S. patent application Ser. No. 10/091,268,filed Mar. 4, 2002, and published Apr. 15, 2003, as U.S. Pat. Pub. No.US 2003/0068320; U.S. patent application Ser. No. 10/091,313, filed Mar.4, 2002, and published Mar. 6, 2003, as U.S. Pat. Pub. No. US2003/0044406; and U.S. patent application Ser. No. 10/657,006, filedSep. 5, 2003, and published Dec. 30, 2004, as U.S. Pat. Pub. No. US2004/0265315, the contents of which are hereby incorporated by referencein their entirety.

5.4.2.5. Antibodies Comprising at Least One Thioether Cross-Link thatSpecifically Bind to CD19 and Compositions Comprising the Same

The present invention provides an isolated antibody that comprises atleast one thioether cross-link and specifically binds to CD19 and acomposition comprising this antibody. The thioether cross-link isdescribed in detail in Section 5.2. The antibodies of the presentinvention can comprise one or multiple thioether cross-links. Thethioether cross-link is described in detail in the section above. Thethioether cross-link can link any two residues of the antibodies. Incertain embodiments, the residues linked by the thioether cross-link arenatural residues. In preferred embodiments, two of the residues arecysteine residues. The thioether cross-link can be at any location ofthe antibodies where feasible according to the knowledge of those ofskill in the art. In preferred embodiments, the thioether cross-linklinks a heavy chain and a light chain of the antibodies. In particularpreferred embodiments, the thioether cross-link links a cysteine of aheavy chain and a cysteine of a light chain of the antibodies.

This antibody of the invention specifically binds to CD19. Theantibodies can be monoclonal antibodies, human antibodies, humanizedantibodies or chimeric antibodies. In some preferred embodiments, theanti-CD19 antibody of the invention is MT-103™. MT-103™ is themost-advanced clinical representative of a novel class of antibodyderivatives called Bi-Specific T Cell Engagers (BiTE™). The BiTEcompound MT-103™ directs and activates the patient's own immune systemagainst the cancer cells, stimulating T cells (a very potent type ofwhite blood cell) to destroy B tumor cells (cancerous white bloodcells). MT-103™ specifically targets a particular protein (the CD19antigen), which is present on cancerous B cells but not on other typesof blood cells or healthy tissues, therefore avoiding the side effectsof traditional chemotherapy

The anti-CD19 antibodies of this section can be made, formulated,administered, used therapeutically or prophylactically, or in othercontext as described in U.S. Pat. No. 6,723,538, and U.S. Pat. Pub. No.2004/0162411, which are incorporated herein by reference in theirentirety. The amino acid sequences of the V_(H) and V_(L) domains ofMT-103™ are listed in Table 1 and provided in U.S. Patent PublicationNo. 2004/0162411.

5.4.2.6. Antibodies Comprising at Least One Thioether Cross-Link thatSpecifically Bind to an Eph Receptor and Compositions Comprising theSame

The present invention provides an isolated antibody that comprises atleast one thioether cross-link and specifically binds to an Eph receptorand a compositions comprising this antibody. The thioether cross-link isdescribed in detail in Section 5.2. The antibodies of the presentinvention can comprise one or multiple thioether cross-links. Thethioether cross-link can link any two residues of the antibody. Incertain embodiments, the residues linked by the thioether cross-link arenatural residues. In preferred embodiments, two of the residues arecysteine residues. The thioether cross-link can be at any location ofthe antibodies where feasible according to the knowledge of those ofskill in the art. In preferred embodiments, the thioether cross-linklinks a heavy chain and a light chain of the antibody. In particularpreferred embodiments, the thioether cross-link links a cysteine of aheavy chain and a cysteine of a light chain of the antibody.

As used herein, the term “Eph receptor” or “Eph receptor tyrosinekinase” refers to any Eph receptor that has or will be identified andrecognized by the Eph Nomenclature Committee (Eph NomenclatureCommittee, 1997, Cell 90:403-404). Eph receptors of the presentinvention include, but are not limited to EphA1, EphA2, EphA3, EphA4,EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB5 and EphB6.In a specific embodiment, an Eph receptor polypeptide is from anyspecies. In a preferred embodiment, an Eph receptor polypeptide ishuman. The nucleotide and/or amino acid sequences of Eph receptorpolypeptides can be found in the literature or public databases (e.g.,GenBank), or the nucleotide and/or amino acid sequences can bedetermined using cloning and sequencing techniques known to one of skillin the art. The GenBank Accession Nos. for the nucleotide and amino acidsequences of the human Eph receptors are summarized in TABLE 2 below.

TABLE 2 Eph Receptor Nucleotide Sequence Amino Acid Sequence EphA1NM_005232.2 NP_005223.2 EphA2 NM_004431.2 NP_004422.2 EphA3, variant 1NM_005233.3 NP_005224.2 EphA3, variant 2 NM_182644.1 NP_872585.1 EphA4NM_004438.3 NP_004429.1 EphA5, variant 1 NM_004439.3 NP_004430.2 EphA5,variant 2 NM_182472.1 NP_872272.1 EphA6 (predicted) XM_114973.4XP_114973.4 EphA7 NM_004440.2 NP_004431.1 EphA8 NM_020526.2 NP_065387.1EphB1 NM_004441.2 NP_004432.1 EphB2, variant 1 NM_017449.1 NP_059145.1EphB2, variant 2 NM_004442.4 NP_004433.2 EphB3 NM_004443.3 NP_004434.2EphB4 NM_004444.3 NP_004435.3 EphB5 (chicken; human NM_001004387.1NP_001004387.1 sequence not reported) EphB6 NM_004445.1 NP_004436.1

In some embodiments, the present invention provides an isolated antibodythat comprises at least one thioether cross-link and specifically bindsto EphA2 and a compositions comprising this antibody. The antibodies ofthe invention can be monoclonal antibodies, human antibodies, humanizedantibodies or chimeric antibodies. In some embodiments, the anti-EphA2antibody of the invention is EA2. In some preferred embodiments, the EA2antibody is human or humanized. In other embodiments, the is EA5. Insome preferred embodiments, the EA5 antibody is human or humanized.Hybridomas producing the anti-EphA2 antibodies of the invention havebeen deposited with the American Type Culture Collection (ATCC, P.O. Box1549, Manassas, Va. 20108) under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedures, and assigned accession numbers, whichare incorporated by reference, as shown in TABLE 3.

TABLE 3 EphA2 Antibodies Deposit No. Date of Deposit EA2.31 PTA-4380 May22, 2002 EA5.12 PTA-4381 May 22, 2002 Eph099B-102.147 PTA-4572 Aug. 7,2002 Eph099B-208.261 PTA-4573 Aug. 7, 2002 Eph099B-210.248 PTA-4574 Aug.7, 2002 Eph099B-233.152 PTA-5194 May 12, 2003 Eph101.530.241 PTA-4724Sep. 26, 2002

EphA2 is a 130 kDa receptor tyrosine kinase that is expressed in adultepithelia, where it is found at low levels and is enriched within sitesof cell-cell adhesion (Zantek, et al, Cell Growth & Differentiation10:629, 1999; Lindberg, et al., Molecular & Cellular Biology 10: 6316,1990). EphA2 is upregulated on a large number of aggressive carcinomacells. The anti-EphA2 antibodies of this invention can be used, forexample, in the treatment of a variety of tumors, including breast,colon, prostate, lung and skin cancers, as well as to preventmetastasis.

The anti-EphA2 antibodies of this section can be made, formulated,administered, used therapeutically or used prophylactically as describedin U.S. patent application Ser. No. 10/823,259, filed Apr. 12, 2004;U.S. patent application Ser. No. 10/823,254, filed on Apr. 12, 2004;U.S. patent application Ser. No. 10/436,782, filed on May 12, 2003 andpublished Feb. 12, 2004 as U.S. Pat. Pub. No. 2004/0028685; U.S. patentapplication Ser. No. 10/436,783, filed on May 12, 2003 and published May13, 2004 as U.S. Pat. Pub. No. 2004/0091486; U.S. patent applicationSer. No. 11/004,794, filed on Dec. 3, 2004; U.S. patent application Ser.No. 10/994,129, filed on Nov. 19, 2004; U.S. patent application Ser. No.11/004,795, filed on Dec. 3, 2004; and U.S. Provisional Application Nos.60/662,517, 60/622,711, 60/622,489, filed Oct. 27, 2004, the contents ofwhich are hereby incorporated by reference in their entirety.

In some embodiments, the antibody comprises the amino acid sequence ofthe V_(H) and V_(L) chains of EA2. In other embodiments, the antibodycomprises the amino acid sequence of the CDRs of the V_(H) and V_(L)chains of EA2. The amino acid sequences of the V_(H) and V_(L) chains ofEA2 and the CDRs of the V_(H) and V_(L) chains of EA2 are listed inTable 1 and are provided in U.S. Patent Publication No. 2004/0028685,published on Feb. 12, 2004, the contents of which are herebyincorporated by reference in its entirety.

The present invention provides an isolated antibody that comprises atleast one thioether cross-link and specifically binds to an antigen ofEphA4 and a compositions comprising this antibody. The antibodies of theinvention can be monoclonal antibodies, human antibodies, humanizedantibodies or chimeric antibodies. Hybridomas producing the anti-EphA4antibodies of the invention have been deposited with the American TypeCulture Collection (ATCC, P.O. Box 1549, Manassas, Va. 20108) on Jun. 4,2004 under the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedures, and assigned accession number PTA-6044 and PTA-4381 andincorporated by reference.

EphA4 is a receptor tyrosine kinase that is expressed in brain, heart,lung, muscle, kidney, placenta, pancreas (Fox, et al, Oncogene 10:897,1995) and melanocytes (Easty, et al., Int. J. Cancer 71:1061, 1997).EphA4 is overexpressed in a number of cancers. The anti-EphA4 antibodiesof this section can be used, for example, to decrease the expression ofEphA4 in the treatment of pancreatic cancers etc.

The anti-EphA4 antibodies of this section can be made, formulated,administered, used therapeutically or used prophylactically as describedin U.S. patent application Ser. No. 10/863,729, filed Jun. 7, 2004; U.S.patent application Ser. No. 11/004,794, filed on Dec. 3, 2004; U.S.patent application Ser. Nos. 11/004,794 and 11/004,795, filed on Dec. 3,2004, the contents of which are hereby incorporated by reference intheir entirety.

The present invention provides an isolated antibody that comprises atleast one thioether cross-link and specifically binds to an antigen ofEphB4 and a compositions comprising this antibody. The antibodies of theinvention can be monoclonal antibodies, human antibodies, humanizedantibodies or chimeric antibodies.

The anti-EphB4 antibodies of this section can be made, formulated,administered, used therapeutically or used prophylactically as describedin U.S. Patent Application Publication Nos. 2003/0207447, 2005/0249736,2005/0164965 and 2005/0084873, International Application Publication No.WO 99/52541, U.S. Pat. Nos. 6,864,227 and 6,579,683, the contents ofwhich are hereby incorporated by reference in their entirety.

5.4.2.7. Antibodies Comprising at Least One Thioether Cross-Link thatSpecifically Bind to IL-9 and Compositions Comprising the Same

The present invention provides an isolated antibody that comprises atleast one thioether cross-link and specifically binds to IL-9 and acompositions comprising this antibody. The thioether cross-link isdescribed in detail in Section 5.2. The antibodies of the presentinvention can comprise one or multiple thioether cross-links. Thethioether cross-link can link any two residues of the antibody. Incertain embodiments, the residues linked by the thioether cross-link arenatural residues. In preferred embodiments, two of the residues arecysteine residues. The thioether cross-link can be at any location ofthe antibodies where feasible according to the knowledge of those ofskill in the art. In preferred embodiments, the thioether cross-linklinks a heavy chain and a light chain of the antibody. In particularpreferred embodiments, the thioether cross-link links a cysteine of aheavy chain and a cysteine of a light chain of the antibody.

This antibody of the invention specifically binds to IL-9. Theantibodies of the invention can be monoclonal antibodies, humanantibodies, humanized antibodies or chimeric antibodies. In somepreferred embodiments, the anti-IL-9 antibodies is MEDI-528. In somepreferred embodiments, the anti-IL-9 antibodies is 7F3com-2H2.

In some embodiments, the antibody comprises the amino acid sequence ofthe V_(H) and V_(L) chains of 7F3com-2H2. In other embodiments, theantibody comprises the amino acid sequence of the CDRs of the V_(H) andV_(L) chains of 7F3com-2H2. The amino acid sequences of the CDRs of theV_(H) and V_(L) chains of 7F3com-2H2 are listed in Table 1 and areprovided in U.S. Patent Publication No. 2005/002934, published on Jan.16, 2005, the contents of which are hereby incorporated by reference inits entirety.

It has been shown that IL-9 may be a key mediator of asthma and may alsocontribute to other respiratory disorders including chronic obstructivepulmonary disease (COPD) and cystic fibrosis. The anti-IL-9 antibodiesof this section may be used in the prophylaxis or treatment of asthma.

The anti-IL-9 antibodies of this section can be made, formulated,administered, used therapeutically or used prophylactically as describedin U.S. patent application Ser. No. 10/823,253, filed Apr. 12, 2004 andpublished Jan. 6, 2005, as U.S. Pat. Pub. No. US 2005/0002934 A1; U.S.patent application Ser. No. 10/823,810, filed on Apr. 12, 2004; U.S.Provisional Application Nos. 60/371,728 and 60/371,683, filed Apr. 12,2002; and U.S. Provisional Application No. 60/561,845, filed Apr. 12,2004, the contents of which are hereby incorporated by reference intheir entirety.

5.4.2.8. Antibodies Comprising at Least One Thioether Cross-Link thathave Therapeutic Utility Compositions Comprising the Same

The invention also encompasses antibodies comprising at least onethioether cross-link that have therapeutic utility, including but notlimited to antibodies listed in TABLE 4. These antibody listed in Table3 can be engineered and/or enriched to comprise at least one thioethercross-link.

TABLE 4 THERAPEUTIC ANTIBODIES THAT CAN BE ENGINEERED ACCORDING TO THEMETHODS OF THE INVENTION Company Product Disease Target Abgenix ABX-EGFCancer EGF receptor AltaRex OvaRex ovarian cancer tumor antigen CA125BravaRex metastatic tumor antigen MUC1 cancers Antisoma Theragyn ovariancancer PEM antigen (pemtumomabytrrium- 90) Therex breast cancer PEMantigen Boehringer Blvatuzumab head & neck CD44 Ingelheim cancerCentocor/J&J Panorex Colorectal 17-1A cancer ReoPro PTCA gp IIIb/IIIaReoPro Acute MI gp IIIb/IIIa ReoPro Ischemic stroke gp IIIb/IIIa CorixaBexocar NHL CD20 CRC MAb, idiotypic 105AD7 colorectal cancer gp72Technology vaccine Crucell Anti-EpCAM cancer Ep-CAM Cytoclonal MAb, lungcancer non-small cell NA lung cancer Genentech Herceptin metastaticbreast HER-2 cancer Herceptin early stage HER-2 breast cancer RituxanRelapsed/refractory CD20 low-grade or follicular NHL Rituxanintermediate & CD20 high-grade NHL MAb-VEGF NSCLC, VEGF metastaticMAb-VEGF Colorectal VEGF cancer, metastatic AMD Fab age-related CD18macular degeneration E-26 (2^(nd) gen. IgE) allergic asthma IgE &rhinitis IDEC Zevalin (Rituxan + low grade of CD20 yttrium-90)follicular, relapsed or refractory, CD20-positive, B-cell NHL andRituximab- refractory NHL ImClone Cetuximab + innotecan refractory EGFreceptor colorectal carcinoma Cetuximab + cisplatin & newly diagnosedEGF receptor radiation or recurrent head & neck cancer Cetuximab + newlydiagnosed EGF receptor gemcitabine metastatic pancreatic carcinomaCetuximab + cisplatin + recurrent or EGF receptor 5FU or Taxolmetastatic head & neck cancer Cetuximab + newly diagnosed EGF receptorcarboplatin + paclitaxel non-small cell lung carcinoma Cetuximab +cisplatin head & neck EGF receptor cancer (extensive incurable local-regional disease & distant metasteses) Cetuximab + radiation locallyadvanced EGF receptor head & neck carcinoma BEC2 + Bacillus small celllung mimics ganglioside Calmette Guerin carcinoma GD3 BEC2 + Bacillusmelanoma mimics ganglioside Calmette Guerin GD3 IMC-1C11 colorectalcancer VEGF-receptor with liver metasteses ImmonoGen nuC242-DM1Colorectal, nuC242 gastric, and pancreatic cancer ImmunoMedicsLymphoCide Non-Hodgkins CD22 lymphoma LymphoCide Y-90 Non-Hodgkins CD22lymphoma CEA-Cide metastatic solid CEA tumors CEA-Cide Y-90 metastaticsolid CEA tumors CEA-Scan (Tc-99m- colorectal cancer CEA labeledarcitumomab) (radioimaging) CEA-Scan (Tc-99m- Breast cancer CEA labeledarcitumomab) (radioimaging) CEA-Scan (Tc-99m- lung cancer CEA labeledarcitumomab) (radioimaging) CEA-Scan (Tc-99m- intraoperative CEA labeledarcitumomab) tumors (radio imaging) LeukoScan (Tc-99m- soft tissue CEAlabeled sulesomab) infection (radioimaging) LymphoScan (Tc-99m-lymphomas CD22 labeled) (radioimaging) AFP-Scan (Tc-99m- liver 7gem-cell AFP labeled) cancers (radioimaging) Intracel HumaRAD-HN (+ head& neck NA yttrium-90) cancer HumaSPECT colorectal NA imaging MedarexMDX-101 (CTLA-4) Prostate and CTLA-4 other cancers MDX-210 (her-2Prostate cancer HER-2 overexpression) MDX-210/MAK Cancer HER-2 MedImmuneVitaxin Cancer αvβ₃ Merck KGaA MAb 425 Various cancers EGF receptorIS-IL-2 Various cancers Ep-CAM Millennium Campath chronic CD52(alemtuzumab) lymphocytic leukemia NeoRx CD20-streptavidin (+Non-Hodgkins CD20 biotin-yttrium 90) lymphoma Avidicin (albumin +metastatic NA NRLU13) cancer Peregrine Oncolym (+ iodine-131)Non-Hodgkins HLA-DR 10 beta lymphoma Cotara (+ iodine-131) unresectableDNA-associated malignant proteins glioma Pharmacia C215 (+staphylococcal pancreatic NA Corporation enterotoxin) cancer MAb,lung/kidney lung & kidney NA cancer cancer nacolomab tafenatox colon &NA (C242 + staphylococcal pancreatic enterotoxin) cancer Protein DesignNuvion T cell CD3 Labs malignancies SMART M195 AML CD33 SMART 1D10 NHLHLA-DR antigen Titan CEAVac colorectal CEA cancer, advanced TriGemmetastatic GD2-ganglioside melanoma & small cell lung cancer TriAbmetastatic breast MUC-1 cancer Trilex CEAVac colorectal CEA cancer,advanced TriGem metastatic GD2-ganglioside melanoma & small cell lungcancer TriAb metastatic breast MUC-1 cancer Viventia NovoMAb-G2Non-Hodgkins NA Biotech radiolabeled lymphoma Monopharm C colorectal &SK-1 antigen pancreatic carcinoma GlioMAb-H (+ gelonin gliorna, NAtoxin) melanoma & neuroblastoma Xoma Rituxan Relapsed/refractory CD20low-grade or follicular NHL Rituxan intermediate & CD20 high-grade NHLING-1 adenomcarcinoma Ep-CAM

5.4.2.9. Antibodies Comprising at Least One Thioether Cross-Link thatcan be Used for Inflammatory Disorders or Autoimmune Diseases andCompositions Comprising the Same

The invention further contemplates any of the antibodies known in theart for the treatment and/or prevention of autoimmune disease orinflammatory disease, wherein the antibodies comprise at least onethioether cross-link. A non-limiting example of the antibodies that areused for the treatment or prevention of inflammatory disorders which canbe engineered according to the invention is presented in TABLE 5A, and anon-limiting example of the antibodies that are used for the treatmentor prevention of autoimmune disorder is presented in Table 5B. Theseantibody listed in Table 5A and 5B can be engineered and/or enriched tocomprise at least one thioether cross-link.

TABLE 5A ANTIBODIES FOR INFLAMMATORY DISEASES AND AUTOIMMUNE DISEASESTHAT CAN PRODUCED IN ACCORDANCE WITH THE INVENTION. Antibody TargetProduct Name Antigen Type Isotype Sponsors Indication 5G1.1 ComplementHumanized IgG Alexion Rheumatoid (C5) Pharm Inc Arthritis 5G1.1Complement Humanized IgG Alexion SLE (C5) Pharm Inc 5G1.1 ComplementHumanized IgG Alexion Nephritis (C5) Pharm Inc 5G1.1-SC ComplementHumanized ScFv Alexion Cardiopulmonary (C5) Pharm Inc Bypass 5G1.1-SCComplement Humanized ScFv Alexion Myocardial (C5) Pharm Inc Infarction5G1.1-SC Complement Humanized ScFv Alexion Angioplasty (C5) Pharm IncABX-CBL CBL Human Abgenix Inc GvHD ABX-CBL CD147 Murine IgG Abgenix IncAllograft rejection ABX-IL8 IL-8 Human IgG2 Abgenix Inc PsoriasisAntegren VLA-4 Humanized IgG Athena/Elan Multiple Sclerosis Anti- CD11aHumanized IgG1 Genentech Psoriasis CD11a Inc/Xoma Anti- CD18 HumanizedFab′2 Genentech Inc Myocardial CD18 infarction Anti- CD18 Murine Fab′2Pasteur- Allograft rejection LFA1 Merieux/ Immunotech Antova CD40LHumanized IgG Biogen Allograft rejection Antova CD40L Humanized IgGBiogen SLE BTI-322 CD2 Rat IgG Medimmune GvHD, Psoriasis Inc CDP571TNF-alpha Humanized IgG4 Celltech Crohn's CDP571 TNF-alpha HumanizedIgG4 Celltech Rheumatoid Arthritis CDP850 E-selectin Humanized CelltechPsoriasis Corsevin M Fact VII Chimeric Centocor Anticoagulant D2E7TNF-alpha Human CAT/BASF Rheumatoid Arthritis Hu23F2G CD11/18 HumanizedICOS Pharm Multiple Sclerosis Inc Hu23F2G CD11/18 Humanized IgG ICOSPharm Stroke Inc IC14 CD14 ICOS Pharm Toxic shock Inc ICM3 ICAM-3Humanized ICOS Pharm Psoriasis Inc IDEC-114 CD80 Primatised IDECPsoriasis Pharm/Mitsubishi IDEC-131 CD40L Humanized IDEC SLE Pharm/EisaiIDEC-131 CD40L Humanized IDEC Multiple Sclerosis Pharm/Eisai IDEC-151CD4 Primatised IgG1 IDEC Rheumatoid Pharm/Glaxo Arthritis SmithKlineIDEC-152 CD23 Primatised IDEC Pharm Asthma/Allergy Infliximab TNF-alphaChimeric IgG1 Centocor Rheumatoid Arthritis Infliximab TNF-alphaChimeric IgG1 Centocor Crohn's LDP-01 beta2- Humanized IgG MillenniumStroke integrin Inc (LeukoSite Inc.) LDP-01 beta2- Humanized IgGMillennium Allograft rejection integrin Inc (LeukoSite Inc.) LDP-02alpha4beta7 Humanized Millennium Ulcerative Colitis Inc (LeukoSite Inc.)MAK- TNF alpha Murine Fab′2 Knoll Pharm, Toxic shock 195F BASF MDX-33CD64 (FcR) Human Medarex/Centeon Autoimmune haematogical disorders MDX-CD4 Human IgG Medarex/Eisai/ Rheumatoid CD4 Genmab Arthritis MEDI-507CD2 Humanized Medimmune Psoriasis Inc MEDI-507 CD2 Humanized MedimmuneGvHD Inc OKT4A CD4 Humanized IgG Ortho Biotech Allograft rejectionOrthoClone CD4 Humanized IgG Ortho Biotech Autoimmune OKT4A diseaseOrthoclone/ CD3 Murine mIgG2a Ortho Biotech Allograft rejection anti-CD3OKT3 RepPro/ gpIIbIIIa Chimeric Fab Centocor/Lilly Complications ofAbciximab coronary angioplasty rhuMab- IgE Humanized IgG1Genentech/Novartis/ Asthma/Allergy E25 Tanox Biosystems SB-240563 IL5Humanized GlaxoSmithKline Asthma/Allergy SB-240683 IL-4 HumanizedGlaxoSmithKline Asthma/Allergy SCH55700 IL-5 Humanized Celltech/ScheringAsthma/Allergy Simulect CD25 Chimeric IgG1 Novartis Allograft rejectionPharm SMART CD3 Humanized Protein Autoimmune a-CD3 Design Lab diseaseSMART CD3 Humanized Protein Allograft rejection a-CD3 Design Lab SMARTCD3 Humanized IgG Protein Psoriasis a-CD3 Design Lab Zenapax CD25Humanized IgG1 Protein Allograft rejection Design Lab/Hoffman- La Roche

TABLE 5B ANTIBODIES FOR AUTOIMMUNE DISORDERS THAT CAN BE PRODUCED INACCORDANCE WITH THE INVENTION Antibody Indication Target Antigen ABX-RB2antibody to CBL antigen on T cells, B cells and NK cells fully humanantibody from the Xenomouse 5c8 (Anti CD-40 Phase II trials were haltedin October CD-40 ligand antibody) 1999 examine “adverse events” IDEC 131systemic lupus erythyematous anti CD40 (SLE) humanized IDEC 151rheumatoid arthritis primatized; anti-CD4 IDEC 152 Asthma primatized;anti-CD23 IDEC 114 Psoriasis primatized anti-CD80 MEDI-507 rheumatoidarthritis; multiple anti-CD2 sclerosis Crohn's disease Psoriasis LDP-02(anti-b7 inflammatory bowel disease a4b7 integrin receptor on white mAb)Chron's disease blood cells (leukocytes) ulcerative colitis SMART Anti-autoimmune disorders Anti-Gamma Interferon Gamma Interferon antibodyVerteportin rheumatoid arthritis MDX-33 blood disorders caused bymonoclonal antibody against FcRI autoimmune reactions receptorsIdiopathic Thrombocytopenia Purpurea (ITP) autoimmune hemolytic anemiaMDX-CD4 treat rheumatoid arthritis and other monoclonal antibody againstCD4 autoimmunity receptor molecule VX-497 autoimmune disorders inhibitorof inosine monophosphate multiple sclerosis dehydrogenase rheumatoidarthritis (enzyme needed to make new RNA inflammatory bowel disease andDNA lupus used in production of nucleotides psoriasis needed forlymphocyte proliferation) VX-740 rheumatoid arthritis inhibitor of ICEinterleukin-1 beta (converting enzyme controls pathways leading toaggressive immune response) VX-745 specific to inflammation inhibitor ofP38MAP kinase involved in chemical signalling of mitogen activatedprotein kinase immune response onset and progression of inflammationEnbrel (etanercept) targets TNF (tumor necrosis factor) IL-8 fully humanmonoclonal antibody against IL-8 (interleukin 8) Apogen MP4 recombinantantigen selectively destroys disease associated T-cells inducesapoptosis T-cells eliminated by programmed cell death no longer attackbody's own cells specific apogens target specific T- cells

5.5. PRODUCTION OF POLYPEPTIDES

The macromolecules of the present invention can be produced by anymethod or technique known in the art. For example, polypeptides can bechemically synthesized or recombinantly produced. See e.g., Sambrook etal., 1990. Molecular Cloning: A Laboratory Manual (Cold Spring HarborPress, Cold Spring Harbor, N.Y.).

5.5.1 Methods of Producing Antibodies

The antibodies of the present invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. Briefly,mice can be immunized with an antigen (either the full length protein ora domain thereof, e.g., the extracellular or the ligand binding domain)and once an immune response is detected, e.g., antibodies specific forthe particular antigen are detected in the mouse serum, the mouse spleenis harvested and splenocytes isolated. The splenocytes are then fused bywell known techniques to any suitable myeloma cells, for example cellsfrom cell line SP20 available from the ATCC. Hybridomas are selected andcloned by limited dilution. Hybridoma clones are then assayed by methodsknown in the art for cells that secrete antibodies capable of binding apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by immunizing mice withpositive hybridoma clones.

Accordingly, monoclonal antibodies can be generated by culturing ahybridoma cell secreting an antibody of the invention wherein,preferably, the hybridoma is generated by fusing splenocytes isolatedfrom a mouse immunized with the antigen with myeloma cells and thenscreening the hybridomas resulting from the fusion for hybridoma clonesthat secrete an antibody able to bind the antigen.

Antibody fragments of the present invention may be generated by anytechnique known to those of skill in the art. For example, Fab andF(ab′)2 fragments of the invention may be produced by proteolyticcleavage of immunoglobulin molecules, using enzymes such as papain (toproduce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2fragments contain the variable region, the light chain constant regionand the CH1 domain of the heavy chain. Further, the antibodies of thepresent invention can also be generated using various phage displaymethods known in the art.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues). The DNA encoding the VH and VL domainsare recombined together with an scFv linker by PCR and cloned into aphagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13 and the VH and VL domains are usually recombinantly fused toeither the phage gene III or gene VIII. Phage expressing an antigenbinding domain that binds to an epitope of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Examples of phage display methodsthat can be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50;Ames et al., 1995, J. Immunol. Methods 184:177; Kettleborough et al.,1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9;Burton et al., 1994, Advances in Immunology 57:191-280; InternationalApplication No. PCT/GB91/01134; International Publication Nos. WO90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and5,969,108; each of which is incorporated herein by reference in itsentirety.

Phage may be screened for antigen binding activities. As described inthe above references, after phage selection, the antibody coding regionsfrom the phage can be isolated and used to generate whole antibodies,including human antibodies, or any other desired antigen bindingfragment, and expressed in any desired host, including mammalian cells,insect cells, plant cells, yeast, and bacteria, e.g., as describedbelow. Techniques to recombinantly produce Fab, Fab′ and F(ab′)2fragments can also be employed using methods known in the art such asthose disclosed in International Publication No. WO 92/22324; Mullinaxet al., 1992, BioTechniques 12:864; Sawai et al., 1995, AJRI 34:26; andBetter et al., 1988, Science 240:1041 (said references incorporated byreference in their entireties).

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing a VHconstant region, e.g., the human gamma 4 constant region, and the PCRamplified VL domains can be cloned into vectors expressing a VL constantregion, e.g., human kappa or lambda constant regions. Preferably, thevectors for expressing the VH or VL domains comprise an EF-1α promoter,a secretion signal, a cloning site for the variable domain, constantdomains, and a selection marker such as neomycin. The VH and VL domainsmay also be cloned into one vector expressing the necessary constantregions. The heavy chain conversion vectors and light chain conversionvectors are then co-transfected into cell lines to generate stable ortransient cell lines that express full-length antibodies, e.g., IgG,using techniques known to those of skill in the art.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use human or chimericantibodies. Completely human antibodies are particularly desirable fortherapeutic treatment of human subjects. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then be bred to producehomozygous offspring which express human antibodies. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., International Publication Nos. WO 98/24893, WO 96/34096, and WO96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporatedby reference herein in their entirety. In addition, companies such asAbgenix, Inc. (Fremont, Calif.) and Medarex (Princeton, N.J.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibodyand a human immunoglobulin constant region. Methods for producingchimeric antibodies are known in the art. See, e.g., Morrison, 1985,Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al.,1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 6,311,415,5,807,715, 4,816,567, and 4,816,397, which are incorporated herein byreference in their entirety. Chimeric antibodies comprising one or moreCDRs from a non-human species and framework regions from a humanimmunoglobulin molecule can be produced using a variety of techniquesknown in the art including, for example, CDR-grafting (EP 239,400;International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, PNAS91:969), and chain shuffling (U.S. Pat. No. 5,565,332).

Often, framework residues in the framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323,which are incorporated herein by reference in their entireties.)

A humanized antibody is an antibody or its variant or fragment thereofwhich is capable of binding to a predetermined antigen and whichcomprises a framework region having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. Preferably, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. Ordinarily, the antibody willcontain both the light chain as well as at least the variable domain ofa heavy chain. The antibody also may include the CH1, hinge, CH2, CH3,and CH4 regions of the heavy chain. The humanized antibody can beselected from any class of immunoglobulins, including IgM, IgG, IgD, IgAand IgE, and any isotype, including IgG₁, IgG₂, IgG₃ and IgG₄. Usuallythe constant domain is a complement fixing constant domain where it isdesired that the humanized antibody exhibit cytotoxic activity, and theclass is typically IgG₁. Where such cytotoxic activity is not desirable,the constant domain may be of the IgG₂ class. The humanized antibody maycomprise sequences from more than one class or isotype, and selectingparticular constant domains to optimize desired effector functions iswithin the ordinary skill in the art. The framework and CDR regions of ahumanized antibody need not correspond precisely to the parentalsequences, e.g., the donor CDR or the consensus framework may bemutagenized by substitution, insertion or deletion of at least oneresidue so that the CDR or framework residue at that site does notcorrespond to either the consensus or the import antibody. Suchmutations, however, will not be extensive. Usually, at least 75% of thehumanized antibody residues will correspond to those of the parentalframework region (FR) and CDR sequences, more often 90%, and mostpreferably greater than 95%. Humanized antibodies can be produced usingvariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239,400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498;Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska etal., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332),and techniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,5,585,089, International Publication No. WO 9317105, Tan et al., 2002,J. Immunol. 169:1119-25, Caldas et al., 2000, Protein Eng. 13:353-60,Morea et al., 2000, Methods 20:267-79, Baca et al., 1997, J. Biol. Chem.272:10678-84, Roguska et al., 1996, Protein Eng. 9:895-904, Couto etal., 1995, Cancer Res. 55 (23 Supp):5973s-5977s, Couto et al., 1995,Cancer Res. 55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et al.,1994, J. Mol. Biol. 235:959-73, Jones et al., 1986, Nature 321:522-525,Riechmann et al., 1988, Nature 332:323, and Presta, 1992, Curr. Op.Struct. Biol. 2:593-596. Often, framework residues in the frameworkregions will be substituted with the corresponding residue from the CDRdonor antibody to alter, preferably improve, antigen binding. Theseframework substitutions are identified by methods well known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework residues important for antigen binding andsequence comparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; andRiechmann et al., 1988, Nature 332:323, which are incorporated herein byreference in their entireties.)

Further, the antibodies of the invention can, in turn, be utilized togenerate anti-idiotype antibodies using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, 1989, FASEB J.7:437-444; and Nissinoff, 1991, J. Immunol. 147:2429-2438). Theinvention provides methods employing the use of polynucleotidescomprising a nucleotide sequence encoding an antibody of the inventionor a fragment thereof.

5.5.2 Recombinant Expression of an Antibody

Recombinant expression of an antibody of the invention, derivative,analog or fragment thereof, (e.g., a heavy or light chain of an antibodyof the invention or a portion thereof or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably, but not necessarily, containing the heavyor light chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, a heavy orlight chain of an antibody, a heavy or light chain variable domain of anantibody or a portion thereof, or a heavy or light chain CDR, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,International Publication Nos. WO 86/05807 and WO 89/01036; and U.S.Pat. No. 5,122,464) and the variable domain of the antibody may becloned into such a vector for expression of the entire heavy, the entirelight chain, or both the entire heavy and light chains.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention or fragments thereof, or a heavy or light chain thereof,or portion thereof, or a single chain antibody of the invention,operably linked to a heterologous promoter. In embodiments for theexpression of double-chained antibodies, vectors encoding both the heavyand light chains may be co-expressed in the host cell for expression ofthe entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention (see, e.g., U.S. Pat. No.5,807,715). Such host-expression systems represent vehicles by which thecoding sequences of interest may be produced and subsequently purified,but also represent cells which may, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibodymolecule of the invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, NS0, and 3T3 cells) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).Preferably, bacterial cells such as Escherichia coli, and morepreferably, eukaryotic cells, especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45: 101; and Cockett et al., 1990,BioTechnology 8:2). In a specific embodiment, the expression ofnucleotide sequences encoding antibodies or fragments thereof whichimmunospecifically bind to and agonize is regulated by a constitutivepromoter, inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO12:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathione5-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, 1984, PNAS 8 1:355-359). Specific initiation signals may also berequired for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol.153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, NS1, T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O and HsS78Bst cells.

The antibodies comprising at least one thioether cross-link can berecombinantly produced by any cell lines for producing antibodies knownto those skilled in the art. It has been found that it is advantageousto produce the antibodies of the invention in melanoma cells. In certainembodiments, the antibodies of the invention are recombinantly producedin melanoma cells. In some embodiments, the antibodies of the inventionare not recombinantly produced in CHO cell line. In other embodiments,the antibodies of the invention are not recombinantly produced in NS0cell line.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), glutamine synthase, hypoxanthine guaninephosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl.Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy etal., 1980, Cell 22:8-17) genes can be employed in tk−, gs−, hgprt− oraprt− cells, respectively. Also, antimetabolite resistance can be usedas the basis of selection for the following genes: dhfr, which confersresistance to methotrexate (Wigler et al., 1980, PNAS 77:357; O'Hare etal., 1981, PNAS 78:1527); gpt, which confers resistance to mycophenolicacid (Mulligan & Berg, 1981, PNAS 78:2072); neo, which confersresistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy3:87; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573; Mulligan,1993, Science 260:926; and Morgan and Anderson, 1993, Ann. Rev. Biochem.62: 191; May, 1993, TIB TECH 11:155-); and hygro, which confersresistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methodscommonly known in the art of recombinant DNA technology may be routinelyapplied to select the desired recombinant clone, and such methods aredescribed, for example, in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981,J. Mol. Biol. 150:1, which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler,1980, PNAS 77:2197). The coding sequences for the heavy and light chainsmay comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced byrecombinant expression, it may be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies of the present invention or fragments thereof may be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

5.6. METHODS OF PRODUCING/ENRICHING MACROMOLECULES THAT COMPRISE ATLEAST ONE THIOETHER CROSS-LINK AND COMPOSITIONS THEREOF

The present invention provides methods for producing a compositionenriched in a macromolecule, wherein the macromolecule comprises atleast one thioether cross-link. The macromolecule can be produced by anymethod for producing a macromolecule known to those skilled in the art.In certain embodiments, the macromolecule is a polypeptide, which can beproduced by any method for producing a polypeptide known to thoseskilled in the art. In further embodiments, the polypeptide is anantibody, which can be produced by any method for producing an antibodyknown to those skilled in the art, including the methods describedabove. The antibody in the composition can be any antibody comprising atleast one thioether cross-link as described herein.

The present invention also provides a method for increasing the amountof a macromolecule which comprises at least one thioether cross-link ina composition. The macromolecule in the composition can be anymacromolecule comprising at least one thioether cross-link as describedin detail herein.

In some embodiments, the methods of the invention comprise incubatingthe composition at a temperature greater than 4° C. for a timesufficient to enrich the macromolecule that comprises at least onethioether cross-link. In certain embodiments, the composition isincubated at room temperature. In certain embodiments, the compositionis incubated at a temperature greater than 10° C. In certainembodiments, the composition is incubated at a temperature greater than15° C. In certain embodiments, the composition is incubated at atemperature greater than 20° C. In certain embodiments, the compositionis incubated at a temperature greater than 30° C. In certainembodiments, the composition is incubated at a temperature greater than37° C. In certain embodiments, the composition is incubated at atemperature between 4° C. and 40° C., between 4° C. and 37° C., between4° C. and 30° C., between 15° C. and 40° C., between 20° C. and 37° C.,between 30° C. and 37° C., or between 37° C. and 40° C.

In some further embodiments, the composition is incubated at atemperature greater than 37° C. for more than three minutes. In somefurther embodiments, the composition is incubated at a temperature about37° C. for more than one day. In some further embodiments, thecomposition is incubated at a temperature about 37° C. for between threeminutes to one day, between three minutes to one month, or between oneday to one month.

In certain embodiments, the composition is incubated at a temperatureabout 40° C. In some further embodiments, the composition is incubatedat a temperature about 40° C. for more than three minutes. In somefurther embodiments, the composition is incubated at a temperature about40° C. for more than one day. In some further embodiments, thecomposition is incubated at a temperature about 40° C. for more than onemonth. In some further embodiments, the composition is incubated at atemperature about 40° C. for between three minutes to one day, betweenthree minutes to one month, or between one day to one month.

In another embodiments, the methods of the invention comprise incubatingthe composition at pH greater than 7 for a time sufficient to enrich themacromolecule that comprises at least one thioether cross-link. Incertain embodiments, the methods of the invention comprise incubatingthe composition at a pH greater than 8. In some further embodiments, themethods of the invention comprise incubating the composition at a pHgreater than 9. In another embodiments, the methods of the inventioncomprise incubating the composition at a pH greater than 10. In certainembodiments, the methods of the invention comprise incubating thecomposition at a pH between 7 and 10, between 7 and 9, between 8 and 9,and between 8 and 10. In certain embodiments, the methods of theinvention comprise incubating the composition at a pH greater than 7 formore than three minutes. In some further embodiments, the methods of theinvention comprise incubating the composition at a pH greater than 7 formore than one hour. In some further embodiments, the methods of theinvention comprise incubating the composition at a pH greater than 7 forbetween three minutes and one hour, between three minutes and one day,and between one hour and one day.

In another embodiments, the method comprises contacting the compositionwith a reducing agent. The reducing agent can be any reducing agent inconnection with preparation of proteins, as known by those skilled inthe art. In certain embodiments, the reducing agent is selected from thegroup consisting of β-mercaptoethanol (BME), dithiothreitol (DTT), NEM,tris(2-carboxyethyl)phosphine (TCEP), and dithioerythritol (DTE).

In a further embodiments, the method of the invention compriseincubating the composition at a temperature greater than 4° C. and at apH greater than 7 for a time sufficient to enrich the macromolecule thatcomprises at least one thioether cross-link.

In another embodiments, the method of invention comprises incubating thecomposition at a temperature greater than 4° C. for a time sufficient toenrich the macromolecule that comprises at least one thioethercross-link and contacting the composition with a reducing agent.

In another embodiments, the method of invention comprises incubating thecomposition at a pH greater than 7 for a time sufficient to enrich themacromolecule that comprises at least one thioether cross-link andcontacting the composition with a reducing agent.

The invention provides methods for producing macromolecules comprisingat one thioether cross-link, the methods comprising incubating themacromolecules in a buffer component such as phosphate buffer or ananalogous buffer. The invention also provides methods for enriching formacromolecules comprising at least one thioether cross-link, the methodscomprising incubating the macromolecules in a buffer component such asphosphate buffer or an analogous buffer. In certain embodiments, thebuffer component is phosphate buffer. In specific embodiments, thebuffer component is not a His buffer. In accordance with the invention,the incubation may be performed at the pHs and/or temperatures disclosedsupra. Further, in certain embodiments, the macromolecule may becontacted with a denaturing reagent such as described above.

In certain embodiments, the present invention provides methods ofisolating a macromolecule comprising a thioether cross-link. Themacromolecule comprising the thioether cross-link can be preparedaccording to any of the methods described above. The macromolecule canbe isolated by any method for purifying the macromolecule apparent toone of skill in the art. For instance, a polypeptide comprising athioether cross-link can be purified according to standard polypeptidepurification techniques apparent to those of skill in the art. Suchtechniques include, but are not limited to, chromatography, ion exchangechromatography, size exclusion chromatography and affinitychromatography.

In certain embodiments, macromolecules comprising thioether cross-linkscan be purified from macromolecules that do not comprise thioethercross-links under denaturing conditions. Useful techniques include gelelectrophoresis, SDS-PAGE and capillary gel electrophoresis. Thepurified macromolecules comprising thioether cross-links can berenatured according to techniques known to those of skill in the art.Advantageously, the stability of the thioether cross-link can permitmore vigorous denaturing and renaturing than what would be tolerated bymacromolecules comprising disulfide bonds.

In further embodiments, macromolecules comprising thioether cross-linkscan be purified from macromolecules that do not comprise thioethercross-links under native conditions. The purification can be accordingto any method of purification apparent to those of skill in the art. Forinstance, certain compositions comprising macromolecules that comprisethioether cross-links and macromolecules that do not comprise thioethercross-links can be incubated under reducing conditions. Reduction of themacromolecules that do not comprise thioether cross-links can lead tochanges in quaternary or tertiary structure that permit resolution ofthe macromolecules that comprise thioether cross-links from themacromolecules that do not comprise thioether cross-links. In furtherembodiments, macromolecules that comprise thioether cross-links can bepurified by affinity chromatography specific for the thioethercross-link. For instance, in certain embodiments antibodies specific fora thioether cross-link bond can be used to purify a macromolecules thatcomprises a thioether cross-link according to antibody affinitypurification techniques known to those of skill in the art. Antibodiesspecific for a thioether cross-link bond can be prepared according tostandard immunological techniques using, for instance, immunogenicmolecules comprising a thioether cross-link. Such immunogenic molecules,for instance, peptides or polypeptides, can be prepared according to thetechniques described herein.

In further embodiments, macromolecules comprising thioether cross-linkscan be prepared synthetically or semi-synthetically according totechniques apparent to those of skill in the art. For instance, peptidesor polypeptides comprising thioether cross-links can be prepared bystandard solution or solid phase synthetic techniques. Activatedprecursor residues comprising thioether cross-links can be preparedaccording to synthetic techniques apparent to those of skill in the art.The peptides or polypeptides can be used themselves or incorporated intolarger macromolecules according to techniques known to those of skill inthe art.

5.7. METHODS OF DECREASING THE AMOUNT OF MACROMOLECULES THAT COMPRISE ATLEAST ONE THIOETHER CROSS-LINK

The present invention further provides a method for decreasing theamount of an antibody which comprises at least one thioether cross-linkin a composition resulting from a first purification method. In certainembodiments, the method comprises carrying out a second purificationmethod identical to said first purification method except that at leastone step of said second purification method is carried out at a lowertemperature and pH than the corresponding step in said firstpurification method, wherein said second purification method results ina lower level of said antibody species than said first purificationmethod. The purification method can be any purification method forantibodies known in the art. Exemplary purification methods include, butare not limited to, chromatography, ion exchange chromatography, sizeexclusion chromatography and affinity chromatography.

5.8. USE OF THE ANTIBODIES AND COMPOSITIONS OF THE PRESENT INVENTION

The present invention provides macromolecules comprising at least onethioether cross-link and compositions comprising the macromolecules ofthe present invention. The macromolecules and compositions of thepresent invention can be used in any context that those of skilled inthe art recognize, for example, diagnosis or therapy etc.

The present invention provides isolated antibodies comprising at leastone thioether cross-link and compositions comprising the antibodies ofthe present invention. The antibodies and compositions of the presentinvention can be used in any context that those of skilled in the artrecognize. For example, the antibodies and compositions of the inventioncan be used directly against a particular antigen. The antibodies andcompositions of the invention can also be used in diagnostic assayseither in vivo or in vitro for detection/identification of theexpression of an antigen in a subject or a biological sample (e.g.,cells or tissues). The antibodies and compositions of the presentinvention can be used alone or in combination with other therapies fortreating, managing, preventing or ameliorating a disorder or one or moresymptoms thereof.

The present invention provides a fusion protein and a compositioncomprising the same, wherein the fusion protein comprises an Fc domainof an antibody or a fragment thereof, and wherein the Fc domain or Fcdomain fragment comprises at least one thioether cross-link. The presentinvention also provides a fusion protein and a composition comprises anC_(H)1, C_(H)2, C_(H)3 and/or C_(L) domain of an antibody, and whereinthe C_(H)1, C_(H)2, C_(H)3 and/or C_(L) domain comprises at least onethioether cross-link. In certain embodiments, the fusion proteincomprises two, three or all of the domains of C_(H)1, C_(H)2, C_(H)3 orC_(L). The fusion protein and compositions of the present invention canbe used in any context that those of skilled in the art recognize, suchas diagnosis or therapy etc.

The present invention provides methods for preventing, managing,treating, or ameliorating a disorder comprising administering to asubject in need thereof one or more antibodies of the invention alone orin combination with one or more therapies (e.g., one or moreprophylactic or therapeutic agents) other than an antibody of theinvention. The present invention also provides compositions comprisingone or more antibodies of the invention and one or more prophylactic ortherapeutic agents other than antibodies of the invention and methods ofpreventing, managing, treating, or ameliorating a disorder or one ormore symptoms thereof utilizing said compositions. Therapeutic orprophylactic agents include, but are not limited to, small molecules,synthetic drugs, peptides, polypeptides, proteins, nucleic acids (e.g.,DNA and RNA nucleotides including, but not limited to, antisensenucleotide sequences, triple helices, RNAi, and nucleotide sequencesencoding biologically active proteins, polypeptides or peptides)antibodies, synthetic or natural inorganic molecules, mimetic agents,and synthetic or natural organic molecules.

Any therapy which is known to be useful, or which has been used or iscurrently being used for the prevention, management, treatment, oramelioration of a disorder or one or more symptoms thereof can be usedin combination with an antibody or a composition of the invention inaccordance with the invention described herein. See, e.g., Gilman etal., Goodman and Gilman's: The Pharmacological Basis of Therapeutics,10th ed., McGraw-Hill, New York, 2001; The Merck Manual of Diagnosis andTherapy, Berkow, M. D. et al. (eds.), 17th Ed., Merck Sharp & DohmeResearch Laboratories, Rahway, N.J., 1999; Cecil Textbook of Medicine,20th Ed., Bennett and Plum (eds.), W. B. Saunders, Philadelphia, 1996for information regarding therapies (e.g., prophylactic or therapeuticagents) which have been or are currently being used for preventing,treating, managing, or ameliorating a disorder or one or more symptomsthereof. Examples of such therapies include, but are not limited to,immunomodulatory agents, anti-inflammatory agents (e.g.,adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide,flunisolide, fluticasone, triamcinolone, methlyprednisolone,prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids,non-steriodal anti-inflammatory drugs (e.g., aspirin, ibuprofen,diclofenac, and COX-2 inhibitors), anti-cancer agents, pain relievers,leukotreine antagonists (e.g., montelukast, methyl xanthines,zafirlukast, and zileuton), beta2-agonists (e.g., albuterol, biterol,fenoterol, isoetharie, metaproterenol, pirbuterol, salbutamol,terbutalin formoterol, salmeterol, and salbutamol terbutaline),anticholinergic agents (e.g., ipratropium bromide and oxitropiumbromide), sulphasalazine, penicillamine, dapsone, antihistamines,anti-malarial agents (e.g., hydroxychloroquine), anti-viral agents, andantibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,erythomycin, penicillin, mithramycin, and anthramycin (AMC)).

In a specific embodiment, the present invention provides administeringone or more humanized anti-RSV antibodies to a subject, preferably ahuman subject, for preventing, treating, managing, or ameliorating a RSVinfection or one or more symptoms thereof in a subject. In oneembodiment, the invention encompasses a method of preventing, treating,managing, or ameliorating a RSV infection or one or more symptomsthereof, said method comprising administering to a subject in needthereof a dose of a prophylactically or therapeutically effective amountof one or more humanized anti-RSV antibodies. In another embodiment, theinvention provides a method of preventing, treating, managing, orameliorating a RSV infection or one or more symptoms thereof, saidmethod comprising administering a prophylactically or therapeuticeffective amount of one or more humanized anti-RSV antibodies andanother therapy.

In a specific embodiment, the present invention provides administeringone or more humanized anti-CD2 antibodies to a subject, preferably ahuman subject, for preventing, treating, managing, or ameliorating Tcell lymphoma related conditions or one or more symptoms thereof in asubject. In one embodiment, the invention encompasses a method ofpreventing, treating, managing, or ameliorating T cell lymphoma relatedconditions or one or more symptoms thereof, said method comprisingadministering to a subject in need thereof a dose of a prophylacticallyor therapeutically effective amount of one or more humanized anti-CD2antibodies. In another embodiment, the invention provides a method ofpreventing, treating, managing, or ameliorating T cell lymphoma relatedconditions or one or more symptoms thereof, said method comprisingadministering a prophylactically or therapeutic effective amount of oneor more humanized anti-CD2 antibodies and another therapy.

In a specific embodiment, the present invention provides administeringone or more humanized anti-IL-9 antibodies to a subject, preferably ahuman subject, for preventing, treating, managing, or ameliorating arespiratory condition or one or more symptoms thereof.

In one embodiment, the invention encompasses a method of preventing,treating, managing, or ameliorating a respiratory disorder or one ormore symptoms thereof (e.g., an allergy, wheezing, and asthma), saidmethod comprising administering to a subject in need thereof a dose of aprophylactically or therapeutically effective amount of one or morehumanized anti-IL-9 antibodies. In another embodiment, the inventionprovides a method of preventing, treating, managing, or ameliorating arespiratory infection or one or more symptoms thereof, said methodcomprising administering a prophylactically or therapeutic effectiveamount of one or more humanized anti-IL-9 antibodies.

In a specific embodiment, the present invention provides administeringone or more humanized anti-EphA2 antibodies to a subject, preferably ahuman subject, for preventing, treating, managing, or ameliorating ahyperproliferative cell disease or one or more symptoms thereof. In oneembodiment, one or more humanized anti-EphA2 antibodies are administeredalone or in combination with other agents to a subject to prevent,treat, manage, or ameliorate cancer or one or more symptoms thereof(see, e.g., U.S. application Ser. No. 10/436,782, which is incorporatedherein by reference in its entirety). In another embodiment, one or morehumanized anti-EphA2 antibodies are administered alone or in combinationwith other agents to a subject to prevent, treat, manage, or amelioratea disorder involving non-neoplastic hyperproliferative cells, inparticular hyperproliferative epithelial and endothelial cells, or oneor symptoms thereof (see e.g., U.S. Application Ser. No. 60/462,024,which is incorporated herein by reference in its entirety). In yetanother embodiment, one or more humanized anti-EphA2 antibodies are usedfor diagnostic or screening purposes.

In a specific embodiment, the present invention provides administeringone or more humanized anti-EphA4 antibodies to a subject, preferably ahuman subject, for preventing, treating, managing, or ameliorating acancer such as a pancreatic cancer or one or more symptoms thereof in asubject. In one embodiment, the invention encompasses a method ofpreventing, treating, managing, or ameliorating a cancer such as apancreatic cancer or one or more symptoms thereof, said methodcomprising administering to a subject in need thereof a dose of aprophylactically or therapeutically effective amount of one or morehumanized anti-EphA4 antibodies. In another embodiment, the inventionprovides a method of preventing, treating, managing, or ameliorating acancer such as a pancreatic cancer or one or more symptoms thereof, saidmethod comprising administering a prophylactically or therapeuticeffective amount of one or more humanized anti-EphA4 antibodies andanother therapy.

In a specific embodiment, the present invention provides administeringone or more humanized anti-EphB4 antibodies to a subject, preferably ahuman subject, for preventing, treating, managing, or ameliorating acancer or one or more symptoms thereof, or inhibiting angiogenesis in asubject. In one embodiment, the invention encompasses a method ofpreventing, treating, managing, or ameliorating a cancer or one or moresymptoms thereof, or inhibiting angiogenesis, said method comprisingadministering to a subject in need thereof a dose of a prophylacticallyor therapeutically effective amount of one or more humanized anti-EphB4antibodies. In another embodiment, the invention provides a method ofpreventing, treating, managing, or ameliorating a cancer or one or moresymptoms thereof, or inhibiting angiogenesis, said method comprisingadministering a prophylactically or therapeutic effective amount of oneor more humanized anti-EphB4 antibodies and another therapy.

The antibodies and compositions of the invention can be used directlyagainst a particular antigen. In some embodiments, the antibodies andcompositions of the invention belong to a subclass or isotype that iscapable of mediating the lysis of cells to which the antibody binds. Ina specific embodiment, the antibodies of the invention belong to asubclass or isotype that, upon complexing with cell surface proteins,activates serum complement and/or mediates antibody dependent cellularcytotoxicity (ADCC) by activating effector cells such as natural killercells or macrophages.

The biological activities of antibodies are known to be determined, to alarge extent, by the constant domains or Fc region of the antibodymolecule (Uananue and Benacerraf, Textbook of Immunology, 2nd Edition,Williams & Wilkins, p. 218 (1984)). This includes their ability toactivate complement and to mediate antibody-dependent cellularcytotoxicity (ADCC) as effected by leukocytes. Antibodies of differentclasses and subclasses differ in this respect, as do antibodies from thesame subclass but different species; according to the present invention,antibodies of those classes having the desired biological activity areprepared. Preparation of these antibodies involves the selection ofantibody constant domains and their incorporation in the humanizedantibody by known technique. For example, mouse immunoglobulins of theIgG3 and IgG2a class are capable of activating serum complement uponbinding to the target cells which express the cognate antigen, andtherefore humanized antibodies which incorporate IgG3 and IgG2a effectorfunctions are desirable for certain therapeutic applications.

In some embodiments, the antibodies and compositions of this inventionare useful in passively immunizing patients.

The antibodies and compositions of the invention can also be used indiagnostic assays either in vivo or in vitro fordetection/identification of the expression of an antigen in a subject ora biological sample (e.g., cells or tissues). Non-limiting examples ofusing an antibody, or a composition comprising an antibody in adiagnostic assay are given in U.S. Pat. Nos. 6,392,020; 6,156,498;6,136,526; 6,048,528; 6,015,555; 5,833,988; 5,811,310; 8 5,652,114;5,604,126; 5,484,704; 5,346,687; 5,318,892; 5,273,743; 5,182,107;5,122,447; 5,080,883; 5,057,313; 4,910,133; 4,816,402; 4,742,000;4,724,213; 4,724,212; 4,624,846; 4,623,627; 4,618,486; 4,176,174 (all ofwhich are incorporated herein by reference). Suitable diagnostic assaysfor the antigen and its antibodies depend on the particular antibodyused. Non-limiting examples are an ELISA, sandwich assay, and stericinhibition assays. For in vivo diagnostic assays using the antibodies ofthe invention, the antibodies may be conjugated to a label that can bedetected by imaging techniques, such as X-ray, computed tomography (CT),ultrasound, or magnetic resonance imaging (MRI). The antibodies of theinvention can also be used for the affinity purification of the antigenfrom recombinant cell culture or natural sources.

6. EXAMPLES

The following examples are offered to illustrate this invention and notto be construed in any way as limiting the scope of this invention.

6.1. Identification, Isolation, Characterization of Antibodies thatComprise a Thioether Cross-Link and Specifically Bind to an Antigen ofRSV

This example illustrates the identification, isolation, characterizationof antibodies that comprise a thioether cross-link and that specificallybind to an antigen of RSV. Motavizumab is used in this example. Asdiscussed in the sections above, motavizumab is an IgG1 monoclonalantibody produced by recombinant DNA technology that specifically bindsto an epitope in the A antigenic site of the fusion (F) protein of RSV.Motavizumab antibody is a humanized antibody and consists of the CDRregions specific for the targeted antigen and the constant regions of ahuman γ1 heavy chain and κ light chain. The monoclonal antibody has twointer-chain disulfide bonds to link heavy and light chains, and anothertwo inter-chain disulfide bonds at the hinge region.

6.1.1 Identification of the Antibody Comprising a Thioether Cross-Link

6.1.1.1. By Reducing CGE

Reducing Capillary Gel Electrophoresis (rCGE) was performed. Monoclonalantibody (Mab) samples were diluted into sample dilution buffercontaining SDS in the presence of β-mercaptoethanol, heated in a boilingwater bath for 10 min, and cooled prior to injection to a HP 3Dcapillary electrophoresis system (Agilent Technologies, Palo Alto,Calif.) (12-14). Electromigration injection was performed at −10.0 kVfor 40 sec. The separation took place in an electric field of 390 V/cmfor 22 min at 50° C. in a Hewlett-Packard extended light path fusedsilica capillary (50 μm I.D., 38.5 cm total length, 30 cm effectivelength). Detection was at 220 nm. Molecular weight markers were alsoanalyzed: lysozyme (144,000 kDa), trypsin inhibitor (21,500 Da),carbonic anhydrase (31,000 Da), ovalbumin (45,000 Da), serum albumin(66,200 Da), phosphorylase B (97,000 Da), β-galactosidase (116,000 Da),and myosin (200,000 Da).

CGE utilizes a proprietary polymer solution that creates a dynamicsieving effect that is analogous to the cross-linked polyacrylamidesieving used in conventional slab gel electrophoresis. Both CGE andSDS-PAGE gel systems provide similar information such as fragmentationor aggregation events in the monoclonal antibody by calibrating thesystem with molecular weight markers. However, the results are morereproducible and easier to quantify when a monoclonal antibody isanalyzed by reducing CGE (12-14, 18). rCGE produces a peak pattern thatcorrelates to the migration times of the heavy and light chain of themonoclonal antibody. Three major bands are expected upon analysis of anMab by rCGE: bands representing the heavy chain, the light chain, andthe internal standard (benzoic acid). Analysis of a Mab by rCGE is shownin FIG. 1A. Bands (peaks) were seen at 5.6 min, 11 min, and 13 mincorresponding to the internal standard, light chain, and heavy chain,respectively. In addition, an unexpected band was observed at 14.5 mincorresponding to an apparent molecular weight of 92 kDa based on themolecular weight markers (not shown). This additional band was usuallypresent at the 0.5-2% level of total protein.

6.1.2 By SDS-PAGE

The monoclonal antibody was also analyzed by reducing SDS-PAGE (FIG. 1B)followed by western blotting to verify that the additional band wasproduct-related.

In SDA-PAGE analysis, Mab samples were diluted into sample dilutionbuffer containing 2% SDS in the presence or absence of 5%2-mercaptoethanol. The resulting samples were heated to 80° C. for 10min, cooled, and separated on a 4-20% polyacrylamide gradient gel(Novex) at 2 μg of protein per lane (15). The gels were then stainedwith Coomassie Blue, destained, and densitometry was performed.Molecular weight markers used were: myosin (200,000 Da), β-galactosidase(116,000 Da), phosphorylase b (97,400 Da), bovine serum albumin (66,200Da), ovalbumin (45,000 Da), carbonic anhydrase (31,000 Da), soybeantrypsin inhibitor (21,500 Da), lysozyme (14,400 Da), and aprotinin(6,500 Da).

Western blot was performed. The SDS-PAGE gel was transferred to apolyvinylidene difluoride (PVDF) membrane (Pierce) by electroblotting(constant current 250 mA/blot) for 30 min in 25 mM Tris, 192 mM glycineand 20% methanol solution (16). The PVDF membrane was blocked for 1 hrat ambient temperature in 1×Tris-buffered saline (Bio-Rad, Hercules,Calif.) and 0.05% Tween-20 (TBST). The blot was incubated with biotinlabeled light chain or heavy chain specific IgG (Kirkegaard & Perry,Gaithersburg, Md.) at a 1:1000 dilution for 2 hrs. The membrane waswashed 3× with TBST, and then incubated with streptavidin and alkalinephosphatase (Pierce) at 1:5000 for 2 hrs at ambient temperature (17).Alkaline phosphatase activity was revealed by5-bromo-4-chloro-3′-indolyphosphosphate p-toluidine salt/nitro-bluetetrazolium chloride (BCIP/NBT) development solution (Kirkegaard &Perry).

Similar to the results obtained from the rCGE, an additional band withan apparent molecular weight of 92 kDa was observed on the reducedSDS-PAGE gel in addition to the heavy and light chains (FIG. 1B). The 92kDa band was further evaluated by western blotting, and shown to beimmunoreactive to both heavy and light chain specific antibodies. Whenmore powerful reducing agents were used (e.g., DTT, NEM), no change inthe 9d2 kDa band was seen. The results indicated that the 92 kDa bandmay be a cross-linked species of the heavy and light chains (FIG. 1C).

6.1.3 Separation of Antibody Cross-Linked Species by Size ExclusionChromatography (SEC)

To further investigate the nature of the 92 kDa band observed in rCGEand reducing SDS-PAGE, the heavy-light (H-L) species was separated fordetailed characterization by SEC.

Size exclusion chromatography (SEC) was performed, Mab samples (53mg/mL, 10 mg) were mixed with 630 μL of 8 M guanidine hydrochloride and130 mM Tris (pH 7.6), 40 μL of water and 120 μL of 500 mM DTT andincubated at 37° C. for 1 hr. Neat 4-vinylpyridine (20 uL) was added tothe sample and the incubation continued for 1 hr at ambient temperaturein the dark. The reduced and alkylated antibody was dialyzed in 500 mLof 6 M guanidine hydrochloride for 2 hrs at 4° C. using a 10,000 MWCOdialysis cassette, and injected onto a size exclusion G3000 SW_(XL)column (5 μm, 300 Å, 7.8×300 mm, TosoHaas). The mobile phase was 6 Mguanidine hydrochloride and 1 M phosphate (pH 6.8), runningisocratically at a flow rate of 0.5 mL/min. The early-eluting fractionbefore the heavy and light chain peaks was detected by UV absorbance at280 nm and collected for further characterization. The collected SECfraction was concentrated using a Microcon-YM-30 filter unit andre-injected onto the TosoHaas G3000 SW_(XL) column to verify identityand purity. Separation of Cross-linked Species by Size ExclusionChromatography

As indicated by rCGE, the protein species was present at a low level andcould only be separated after the antibody was reduced; thus, it waschallenging to obtain enough material for characterization. SEC was themethod of choice to collect the H-L species. The denatured, reduced andalkylated monoclonal antibody was separated by SEC using a mobile phasecontaining 6 M guanidine to keep the reduced heavy and light chains,including the modified species, from precipitating and aggregating (FIG.2). The heavy and light chain peaks were detected at 12.7 min and 15.4min, respectively, and an additional peak was observed 12 min. Thisearly-eluting peak was present at 2.1% level, while the heavy chain waspresent at 75.4% and the light chain at 22.4%. The early-eluting peakshould have a higher molecular weight than the heavy and light chainsbecause higher MW compounds pass through the column faster. Theearly-eluting peak was collected for further characterization.

6.1.4 Characterization of the Antibody Comprising a Thioether Cross-Linkfrom SEC Purification

The early-eluting peak in the SEC profile was analyzed by reducingSDS-PAGE, western blot, LC-MS, and tryptic peptide mapping with tandemmass spectrometry. The SEC fraction showed a band with an apparentmolecular weight of 92 kDa by reducing SDS-PAGE. Upon western blot, thisband was reactive to both heavy and light chain specific detectionantibodies. This data indicated that the SEC fraction was thecross-linked species observed by rCGE and reducing SDS-PAGE.

6.1.4.1. Molecular Weight Determination by LC-ESI-MS

The Fab fragment of the antibody was prepared. Mab (1 mg) was digestedwith papain (Sigma) at an enzyme to protein ratio of 1:100 in 500 μL ofphosphate buffer, pH 7.1 in the presence of 2.45 mg/mL cysteine at 37°C. for 1 hr. The papain digest was loaded onto a HiTrap NHS-activated HPaffinity column coupled with Protein L using 2 mM phosphate, pH 7.2. TheFab portion bound to the Protein L column was eluted by 100 mMphosphate, pH 2.0, and collected, neutralized with 2 M Tris, pH 10, andconcentrated with a 30,000 MWCO Microcon filter unit.

On-line LC-MS analysis of the SEC fraction and Fab fragment wasperformed using an Agilent 1100 HPLC system (Agilent Technologies, PaloAlto, Calif.) and ThermoElectron LCQDuo or LTQ ion trap massspectrometer (Thermo Electron, San Jose, Calif.). The SEC fraction wasanalyzed on a reversed phase C18 column (Jupiter, 5 μm, 300 Å, 2×250 mm,Phenomenex) connected to a UV detector followed by the LCQ massspectrometer The Fab fragment was analyzed on a Poroshell reversed phase300-SB-C3 column (5 μm 300 Å, 2.1×75 mm, Agilent Technologies) followedby the LTQ mass spectrometer. The HPLC system used two different mobilephases: mobile phase A (0.1% TFA in water) and mobile phase B (0.1% TFAin acetonitrile). The samples were separated using a linear gradient ofmobile phase B with a flow rate of 200 μL/min. The eluted proteins weremonitored by UV detection at 220 nm and directed to the massspectrometer, which was operated in positive ion mode. The deconvolutionprogram of Bioworks version 3.1 (Thermo Electron) was used todeconvolute the mass spectra to obtain the molecular masses of theproteins.

The actual molecular weight of the early-eluting peak in the SEC profilewas determined by LC-ESI-MS analysis. After mass spectrum deconvolution,a molecular weight of 75,860 Da was obtained, which is consistent withthe combined molecular weights of the alkylated heavy and light chains.This result proves that the 92 kDa band observed in the rCGE andreducing SDS-PAGE is a cross-linked species containing one heavy and onelight chain with an actual molecular weight of 75 kDa. It has beenreported in the literature that during SDS-PAGE analysis, ahalf-antibody molecule migrates at 64 kDa with no heating, but migratesat 92 kDa with heating in SDS. (9). This phenomenon may be explained bythe work of Pitt-Rivers and Impiombato (19), who demonstrated thatproteins bind to 90-100% of their weight of SDS under native conditions,but reduction of all the disulfide bonds present results in more bindingof SDS (up to 1.4 times the weight of the protein). It agrees with theobservation that the apparent MW of the half-antibody (after heating) onthe reducing SDS-PAGE is higher than its actual MW.

6.1.4.2. Peptide Mapping with Tandem Mass Spectrometry

To further investigate the nature of the cross-link modification, theSEC fraction was digested and analyzed by tryptic peptide mapping withtandem mass spectrometry.

Two different procedures were used for peptide mapping: tryptic peptidemapping for reduced samples, and Lys-C peptide mapping for non-reducedFab samples. In the tryptic peptide mapping procedure, the reduced andalkylated antibody and the concentrated SEC fraction of interest wereeach dialyzed into 6 M urea containing 100 mM Tris buffer (pH 8.0) using10,000 MWCO dialysis cassettes for 2 h at 4° C. The dialyzed fractionwas mixed with 50 mM Tris buffer (pH 8.1) in the ratio of sample/Trisbuffer 1:2 v/v. Trypsin (1 μg/μL trypsin in 1 mM HCl) was immediatelyadded at a protein substrate to enzyme ratio of 25:1. The digestionproceeded for 4-5 hrs at 37° C. and was quenched by addingtrifluoroacetic acid (TFA) to a final TFA concentration of 2%. For theLys-C mapping procedure, the Fab fraction was digested by Lys-C (WakoChemicals) at a protein to enzyme ratio of 10:1 in 5 M urea, 25 mMTris-HCl and 1 mM EDTA, pH 8.5, at 37° C. for 8h.

The on-line LC-MS/MS analysis of tryptic peptides was performed using anAgilent 1100 HPLC system and ThermoElectron LTQ ion trap massspectrometer. The HPLC system was equipped with a Polaris C18-A column(5 μm, 4.6×250 mm, Varian) connected to a UV detector followed by themass spectrometer. The HPLC system used two different mobile phases:mobile phase A (0.02% TFA in water) and mobile phase B (0.02% TFA inacetonitrile.) The peptides were eluted using a gradient of 0-20% mobilephase B over 70 min and 20-36% over 90 min at a flow rate of 0.7 mL/min.The eluted peptides were monitored using two different orthogonaldetection systems: UV detection at 220 nm and LCQ mass spectrometerdetection in positive ion mode. The mass spectrometer was operated indata-dependent “triple play” mode with dynamic exclusion enabled. Inthis mode, the instrument continuously acquired full scan mass spectra(m/z 300-2000). When the signal exceeded a predefined threshold, a highresolution “zoom scan” and an MS/MS scan were acquired.

To obtain the exact mass of the thioether linked and disulfide linkedpeptides, the Lys-C digest was analyzed using ThermoElectron LTQinterfaced with Fourier-transform ion cyclotron resonance massspectrometer (FTMS).

The SEC fraction was digested and analyzed by tryptic peptide mappingwith tandem mass spectrometry. The peptide map profile of theearly-eluting SEC fraction was compared to that of the unfractionatedmonoclonal antibody (FIG. 3). Because the proposed cross-linked speciesis a half-antibody, its peptide map profile should be equivalent to thepeptide map profile of the monoclonal antibody and any differencesshould reveal the modification site. In the peptide map profile of theSEC fraction (FIG. 3A), the tryptic peptides H19, H20, L20 and L19-L20were significantly reduced, while two new peptides with molecularweights of 1228 and 4152, respectively, were observed. The peptides thatdecreased in intensity, H19 (SCDK) and L19-L20 (SFNRGEC), may explainthe observed molecular mass of 1228, which corresponded to across-linked peptide between L19-L20 and

H19, with the loss of 32 Da. The molecular mass of 4152 corresponds to across-linked peptide SFNRGEC-SCDKTHTCPPCPAPELLGGPSVFLFPPKPK (L19-L20 andH19-H20), with the loss of 32 Da. Because the SEC fraction was collectedafter a reduction step, these two cross-linked peptides containednon-reducible linkages. The loss of 32 Da may reflect the loss of onesulfur residue from the disulfide bond between the Cys residue on theheavy chain peptide H19 (SCDK) and the C-terminal Cys residue on thelight chain peptide L20 (GEC), which could explain why the twocross-linked peptides could not be reduced. The mass assignments wereconfirmed by tandem mass spectrometry.

6.1.4.3. Location of the Thioether Linkage Between the Light and HeavyChain

The tandem mass spectrometric analysis of the new tryptic peptidesprovided convincing evidence of the exact location of modification. Thefragment mass spectrum (MS/MS) of the doubly charged ion at m/z 615.6⁺²(L19-L20 and H19) is shown in FIG. 4A. The numbering system used wasdescribed by Roepstorff and Fohlman (20). Masses of the C-terminalfragment y series, y₁ to y₇, are consistent with the fragments from thepeptide

SFNRGEC attached with SCDK by a thioether link (loss of a sulfur fromthe disulfide bond) between the two cysteines. The masses of theN-terminal fragment b series of the peptide SFNRGEC, b₁ to b₆, and thefragment ion series from the peptide SCDK, b^(h) ₁ to b^(h) ₄ and y^(h)₁ to y^(h) ₄, support the thioether link between the two cysteineresidues of SFNRGEC and SCDK. There are also some fragment ionsresulting from the linkage breakdown between two peptides (e₇-SH₂, e^(h)₂-SH₂, e₇, and e^(h) ₂), and the a, c and x ion series in the MS/MSspectra. The MS/MS spectrum of H19-20 and H19-20 also supported thethioether linkage between H19-20 and L19-20 (not shown). Therefore, themodified species of the monoclonal antibody are the heavy and lightchains cross-linked by a non-reducible thioether bond between Cys-223 ofthe heavy chain and the C-terminal Cys residue of the light chain (FIG.1C).

6.1.4.4. Molecular Mass Determination of the Fab Fragment

To further demonstrate that the disulfide bond modification was not anartifact resulting from sample preparation under the gel electrophoresisreducing conditions, the modification site was verified usingnon-reduced conditions. It was found that the 92 kDa band in the rCGEincreased with time during incubation at 40° C. After 5.5 months ofstorage at 40° C., about 8% of the 92 kDa band had formed by rCGEanalysis. To make the analysis easier, a monoclonal antibody stored ateither 4° C. (control) or at 40° C. (heat-stressed) was digested bypapain to generate Fab fragments. The Fab fragments were purified by aProtein L affinity column (specific to the antibody light chain). Themolecular weights of two Fab fragments were then determined by LC-MS(FIG. 5).

The most intense ion in the deconvoluted mass spectra of both thecontrol and stressed monoclonal antibodies (FIG. 5, m/z 47628.9 and47629.4) agreed with the theoretical molecular mass 47626 for the Fabfragment consisting of the intact light chain linked to the heavy chainfragment (1-227) through a disulfide bond. The molecular massmeasurement by LC-MS has a variation of ±3 Da. For the heat-stressedantibody (FIG. 5B), a minor component with an m/z of 47598.4 Da, whichwas 31 Da less than the mass of the major component (m/z at 47629.4 Da)was also observed. This mass was in agreement with a thioether bondlinkage between Cys-213 of the light chain and Cys-233 of the heavychain in the Fab fragment. Based on the ion intensity, the relativepercentage of the thioether-linked Fab (47598.4 Da) is 14.5% for theheat-stressed antibody. Thus, the data supports that the thioetherlinkage existed in the monoclonal antibody under non-reduced conditions.

6.1.5 Confirmation of the Thioether Linkage Under Non-Reduced Conditions

To identify the location of the thioether link, the Fab fragments of thecontrol and heat-stressed antibodies were digested by Lys-C undernon-reducing conditions and analyzed by reversed phase HPLC followed byon-line tandem mass spectrometric analysis on a LTQ ion trap instrument.A peptide with a m/z of 1260.4 Da was identified in the Lys-C digest ofboth control and heated-stressed antibodies, which was in agreement withthe expected disulfide bond-linked peptide SFNRGEC-SCDK (data notshown). This is consistent with the molecular mass of the majorcomponent of the Fab fragments. Another peptide with an m/z at 1228.4 Da(32 Da less) and earlier retention time was identified in the Lys-Cdigest of both the control and heated-stressed antibodies, which was inagreement with the thioether-linked peptide SFNRGEC-SCDK. This isconsistent with the molecular mass of the minor thioether-linked Fab forthe heat-stressed antibody. The relative percent of the thioether-linkedpeptide was dramatically different between the control and heat-stressedantibodies. The thioether linked peptide was 13.6% for heat-stressedantibody, but only 0.4% for the antibody stored at 4° C. In the massspectrum of the intact Fab of the control antibody, no visible ioncorresponding to the thioether-linked Fab was observed, which wasattributed to its low percentage and unit resolution of LTQ in full scanmode.

FIG. 4 shows the direct comparison of the tandem mass spectra of thepeptide SFNRGEC-SCDK linked by a disulfide bond and a thioether bond,which were found in both tryptic and Lys-C digests. The fragment ionsinvolving the thioether linkage, such as y₁-y₇, b₇, y^(h) ₃-y^(h) ₄,b^(h) ₂-b^(h) ₄, c^(h) ₂, and c^(h) ₃, are 32 Da less than thecorresponding ions involving the disulfide bond linkage. On the otherhand, the fragment ions not involving the thioether or disulfidelinkage, such as b₁-b₆ and y^(h) ₂, have the same masses for bothpeptides. For the fragment ions resulting from the linkage breakdownbetween two peptides, the disulfide-linked peptide has two additionalions, e₇+S and e^(h) ₂+S, due to the extra sulfur residue, while theother four fragment ions e₇-SH₂, e^(h) ₂-SH₂, e₇, and e^(h) ₂, arecommon to both disulfide-linked and thioether-linked peptides. Thisdirect comparison further provides the evidence for the thioetherlinkage between the peptides SFNRGEC and SCDK.

This example illustrates that a novel, non-reducible thioether bridgebetween the heavy and light chains of IgG1 monoclonal antibodies is thesource of a band with an apparent molecular weight of 92 kDa in reducingrCGE and SDS-PAGE analysis. The analysis of the SEC fraction and the Fabfragment of a monoclonal antibody conclusively proved that the heavy andlight chains were cross-linked by a non-reducible thioether bond betweenCys-223 of the heavy chain and the C-terminal Cys residue of the lightchain (Cys-213). The data support that the thioether linkage existed inthe non-stressed monoclonal antibody, and its content increased with theduration of incubation at 40° C.

7. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. All publications,patents and patent applications mentioned in this specification areherein incorporated by reference into the specification to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated herein byreference.

1. A composition substantially free of a denaturing reagent comprisingan antibody, wherein said antibody comprises at least one thioethercross-link.
 2. The composition of claim 1, wherein the antibody is morethan 2%, 5%, 10% or 15% of the antibodies in the composition.
 3. Thecomposition of claim 2, wherein the antibody is a monoclonal antibody, ahuman antibody, a chimeric antibody, a humanized antibody, a singlechain antibody, a Fab fragment, a Fab′ fragment or a F(ab)′₂ fragment.4. The composition of claim 2, wherein the antibody comprises multiplethioether cross-links.
 5. The composition of claim 2, wherein thethioether cross-link is in the Fab portion or the Fc portion of theantibody.
 6. The composition of claim 2, wherein the thioethercross-link is intra-molecular or inter-chain.
 7. The composition ofclaim 2, wherein the thioether cross-link links a heavy chain and alight chain of the antibody.
 8. The composition of claim 2, wherein thethioether cross-link links two heavy chains of the antibody.
 9. Thecomposition of claim 2, wherein the thioether cross-link links acysteine residue of the heavy chain and a residue of the light chain.10. The composition of claim 2, wherein the thioether links a residue ofthe heavy chain and a cysteine residue of the light chain of theantibody.
 11. The composition of claim 7, wherein the thioethercross-link links a cysteine residue of the heavy chain and a cysteineresidue of the light chain.
 12. The composition of claim 11, wherein thethioether cross-link links a cysteine residue in the C_(H)1 region ofthe heavy chain and a cysteine residue in the C_(L) region of the lightchain.
 13. The composition of claim 12, wherein the cysteine residue inthe C_(H)1 region of the heavy chain is at the amino acid position 223according to the Kabat numbering system.
 14. The composition of claim12, wherein the cysteine residue in the C_(L) region of the light chainis at the amino acid position 213 according to the Kabat numberingsystem.
 15. The composition of claim 2, wherein the antibodyspecifically binds to a respiratory syncytial virus (RSV) antigen, ahuman metapneumovirus (hMPV) antigen, integrin α_(v)β₃, CD2, CD19, anEph receptor, or to IL-9.
 16. A composition substantially free of adenaturing reagent comprising an antibody wherein said antibodycomprises a lanthionine.
 17. The composition of claim 16, wherein theantibody is more than 2%, 5%, 10%, 15% or 20% of the antibodies in thecomposition.
 18. A method for increasing the amount of an antibody thatcomprises at least one thioether cross-link in a composition, saidmethod comprising incubating the composition at a temperature greaterthan 4° C., and/or at pH greater than 7 for a time sufficient toincrease the amount of said antibody comprising at least one thioethercross-link.
 19. The method of claim 18, wherein the step comprisesincubating the composition at a temperature greater than 37° C.,incubating the composition at a pH greater than 8, and/or contacting thecomposition with a reducing agent.
 20. A method for decreasing theamount of an antibody in a composition, wherein said antibody comprisesat least one thioether cross-link in a composition resulting from afirst purification method, said method comprising carrying out a secondpurification method identical to said first purification method exceptthat at least one step of said second purification method is carried outat a lower temperature and/or lower pH than the corresponding step insaid first purification method, wherein said second purification methodresults in a lower level of said antibody comprising at least onethioether cross-link than said first purification method.